purified water freezing experiment

Instant Freeze Water – Bottle Slam

Sharply knock a bottle of supercooled liquid water on the table and it instantly turns to slushy ice before your eyes.

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You put a plastic bottle of soda pop or water in the freezer for a few minutes to get it ice cold. It’s still a liquid when you take it out to enjoy but the second you twist the cap, the liquid instantly turns to slush! The process is amazing to watch but hard to repeat.  This is a great illustration of how supercooled liquids defy freezing even at temperatures well below their freezing points. You’ll need some ice, salt, and several unopened plastic bottles of purified water to attempt this science demo. It’s cool… below-the-freezing-point cool!

But first… If this happens to work on your very first attempt, go buy a lottery ticket! You have to be patient and understand that all of the measurements of time, ice, water, and rock salt are summaries of what has worked for others. Shoot a video of every attempt you make to document your hits and misses – and plan on several misses, too. When the ice crystals do begin to form in the water as planned, expect to hear lots of spontaneous screams and cheers coming from – you! It really is cool. Just remember: NO glass bottles!

Experiment Videos

Here's What You'll Need

Caution: do not use glass bottles, water: bottled, purified, or distilled (several bottles, refrigerated), large, deep bowl or container, crushed ice, thermometer, adult supervision, let's try it.

Nearly fill the container with ice.

Shove two refrigerated, plastic water bottles deeply into the ice. Keep them close to the center of the bowl but keep each surrounded by and buried in ice as much as possible.

Scatter a generous amount of rock salt all over the surface of the ice.

Insert the thermometer into the ice between the bottles. Monitor the temperature. Over the next half-hour, the temperature will fall slowly. Add ice and salt to the container as needed to keep the bottles buried in it. Watch that thermometer!

The temperature in the bowl needs to drop to 17℉ (-8℃). If the water gets too much colder, it may freeze prematurely.

After the water has been this cold for 10 minutes (and is still a liquid), gently remove a bottle from the ice/salt mixture. Strike the bottle sharply against the table. Ice crystals may immediately form near the top of the bottle and quickly move down through the liquid. Carefully remove the second bottle and twist open the cap. The same instant freezing will likely occur from the top down.

How Does It Work

You used salt and ice to drop the temperature in the chill mixture below the normal freezing point of water. This is called “freezing point depression.” This very cold salt water can be used to cool other water and soda samples below their normal freezing point to discover which of them can be be supercooled. You might also discover which samples freeze at their normal freezing points no matter what.

When water freezes, the molecules come together in a very orderly way and form a crystalline structure. Because of this, water molecules as ice have less energy than water molecules as liquid. That means to go from liquid water to solid water, the molecules have to lose heat energy. In other words, as supercooled water freezes when you tap it or open it, it also warms up the rest of the water. This heating may allow only ten or twenty percent of the water to freeze and that accounts for slush being in the bottle instead of it being a solid chunk. The formation of ice crystals happens very quickly but heat flows slowly in water.

When water is cooled to its freezing point, ice crystals can begin to collect in the water. Like snow flakes, these crystals need something on which to grow and they use microscopic impurities in the water or locations on the bottle to do just that. If you work with really pure water and cool it slowly to produce supercooled water as a liquid, there’s different outcome. When an impurity (e.g. an ice crystal) is added to this supercooled pure water, it speeds up the crystallization process even more. The water instantly freezes solid with no slush in it anywhere. This is called “snap freezing.”

If you supercool soda water or soda pop, there are some other factors to consider. When soda pop is produced, large quantities of additives (like sugar, colors, and flavorings) as well as carbon dioxide (CO 2 ) are pumped into water. These additives are called solutes and when solutes are added to a liquid such as water (the solvent), the freezing point of the water drops. By lowering the freezing point, soda has to reach a much colder temperature than plain water to freeze. The carbon dioxide gas in the soda is maintained only as long as the bottle is kept sealed. When the bottle is opened and you hear that “whoosh” of gas and foam rushing out of the bottle, the concentration of solutes in the water quickly goes down. The freezing point goes up and, without all those solutes, the soda freezes very quickly. Of course, all those bubbles provide places for the ice crystals to begin forming, too. You can test this by tapping a supercooled bottle of soda pop without opening it. Bubbles will form after the tap and freezing will likely occur.

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From Liquid to Solid: How Long Does It Take Water to Freeze?

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You may have seen videos of what looks like an ordinary bottle of cold water hanging out and minding its own watery business until bam! Someone taps it against the table and the whole bottle turns instantly to ice . What is this dark wizardry?

It isn't magic but instead science that causes the bottled water to completely freeze — and some pretty simple science at that. So, how long does it take water to freeze ? Let's find out.

The Mystery of Nucleation

Freezing point fun facts, quick freezing faqs and myth busting.

When any substance changes state — like liquid water changing to solid ice — the process involves nucleation . It’s the anchor that creates the first ice crystal and then promotes the rapid formation of more.

Heterogeneous Nucleation

This happens when there are impurities, like dust, present in the water, providing the necessary nucleus for ice formation in water exposed to freezing temperatures. Ice crystals then form throughout the liquid, turning our water into solid ice over time.

Homogeneous Nucleation

Pure water has no impurities, so without a nucleus to kickstart the freezing process, the water becomes supercooled. This allows the water to freeze faster when exposed to an external nucleus, making the magic of "instant" ice possible.

Water famously becomes completely frozen at 32 degrees Fahrenheit (0 degrees Celsius). But when water is devoid of impurities, like in purified bottled water, the freezing process requires even colder temperatures.

So, if you place bottles of purified water in the cold air of a freezer and leave them a couple of hours, they'll still be liquid because pure water with no nuclei in it freezes at minus 43.6 degrees Fahrenheit (minus 42 degrees Celsius). It's now a supercooled liquid, which does indeed sound super cool.

Let's Make Some Instant Ice!

Ready to freeze water? Grab some water bottles and place them in your freezer. Make sure it's undisturbed for a few hours, getting it to that supercooled state. The exact freezing time? Typically, it takes about two-and-a-half to three hours .

Once the wait is over, remove the bottles with care. Then shake one or whack it on the table.

Anything can act as a nucleus at this point — air bubbles, a slight dent in the bottle. Any little change will be enough to cause homogenous nucleation. Once that disturbance is present, the uniform water molecules will freeze completely and so quickly that it looks instant.

An alternative to the whacking or shaking method is to pour the supercooled water over an ice cube. The cube will serve as the nucleus, and you'll be able to create a little tower of ice as you pour.

Which Freezes Faster, Hot or Cold Water?

An interesting phenomenon known as the Mpemba effect suggests that under certain conditions, hot water freezes faster than cold water. Crazy, right?

Do Different Ice Trays Affect the Freezing Process?

Absolutely! A metal ice cube tray, for instance, might speed up the process of freezing water for solid ice cubes compared to a plastic ice tray, because metal ice cube trays conduct heat (and the lack of it). Oversized ice cube trays, on the other hand, might take longer simply due to the larger volume.

What's the Ideal Water for Instant Ice?

Bottled or purified water is typically best for this icy experiment, but tap water, depending on how treated it is, can sometimes work too.

This article was updated in conjunction with AI technology, then fact-checked and edited by a HowStuffWorks editor.

Please copy/paste the following text to properly cite this HowStuffWorks.com article:

Instant Ice Science Experiment

This science experiment is an exciting experiment to show your child water transforming from liquid to a solid instantaneously!

The Instant Ice experiment shows the transformation from liquid to solid in an instant! When purified water is supercooled (cooled below freezing point), it will instantly turn from a liquid to a solid when it is disturbed. This could be by a jolt to the container or just adding an ice cube to it.

To make it more exciting, your child can create fun ice sculptures while pouring the supercooled water. Since it only takes a few items that you likely have on hand, this is an easy at-home experiment.

How to make the Instant Ice experiment

Supplies you will need.

For the Instant Ice experiment, you’ll need:

• Bottles of purified water
• A freezer with space to lay bottles flat

Before you start

I found that water bottles with harder plastic tended to be easier to handle than softer plastic. I used Dasani water bottles and had a much easier time than with a softer plastic bottle like Zephyrhills.

Instructions

Here is how to do the Instant Ice experiment:

Step 1: Place your water bottle(s) in the freezer on their side

I wanted to have a few water bottles in the freezer, just in case I accidentally messed up on the experiment.

It varies for everyone, but your water bottles will likely need at least 1.5 hours to get ready, likely more. Mine needed about 2.5 hours.

If, by 1.5 hours, your water bottles are not ready, check back every 15-20 minutes.

Optional (but encouraged): I also added a water bottle with tap water in it as a control. Once the tap water bottle froze and the purified water was still liquid, I knew it was ready to go.

Step 2: Carefully open the water bottle

Remember how I mentioned that a simple jolt could ignite the freeze? Since you have to hold the bottle in order to unscrew the cap, you will want to be careful about the amount of pressure you place on the bottle.

Step 3: Pour the supercooled water into the empty container

You won’t have to be as careful with this step.

Step 4: Start the freeze!

Take a piece of ice and simply touch it to the surface of the supercooled water. You won’t have to hold it for long: it should instantly activate the freeze and you will be able to see the water transform to ice!

The ice cube you added will sit on top at this point.

Get your child involved : Let your child touch the ice cube to the top of the water and ignite the freeze. They will feel like they have superpowers!

Step 5: Add water to create ice sculptures

You can do this in either container (the newly-formed ice or the container with ice cubes).

Slowly pour the water out of the water bottle and into these containers to create fun ice sculptures!

Get your child involved : Allow your child free reign over the ice sculptures. Let them get creative! There’s no right or wrong with this step.

Here’s a quick video of creating ice sculptures:

The science behind the Instant Ice science experiment

The Instant Ice experiment showcases the transformation from a liquid to a solid in an instant.

How it works

This experiment studies supercooled water, which is when the water’s temperature falls below freezing but does not actually freeze.

When water is very pure, it is difficult for ice crystals to form because they need what is called a “nucleation point” (the first step in the formation of a new thermodynamic phase) to begin freezing.

When supercooled water is disturbed (by hitting it or introducing a piece of ice, like in our experiment), it instantly turns to ice!

More chemistry experiments to try out with your child

• Fizzing lemons experiment – using lemons and baking soda to make a lemon volcano
• Homemade lava lamp – vinegar and baking soda bubble around in a container of oil
• Magnetic Slime – classic slime, but with an interactive lesson in magnets

FAQ about the Instant Ice Experiment

Does the plastic bottle have to be a harder or softer plastic.

In my opinion, plastic bottles with harder plastic allow you to handle them easier in their supercooled state than a softer plastic bottle. When I used a softer plastic bottle, I initiated the freeze accidentally every time.

Can you make instant ice with tap water?

For this experiment, it is not recommended to use regular tap water. Tap water holds contaminants that could be enough for a nucleation point, which would trigger the freeze when the water reaches the freezing point. By using purified water, you have no contaminants, which will allow your water to stay a liquid well under freezing temperatures.

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Level of Education

Post Secondary

Recommended Age

Time Required

• ~10 minutes
• ~20 minutes
• ~30 minutes
• ~45 minutes

1 day or more

Number of people

• 100 – 200 €

Supervision

Instant-Freeze Water

Meta Description

Learning Objectives

Understand how the process of supercooling works and why some liquids do not freeze when cooled below their freezing point. Understand the concept of latent heat of fusion. Introduction to the concept of ice being formed by a lattice structure of water molecules.

Crystallisation The formation of a solid in which the molecules form a highly organised and symmetric structures called crystals.

Crystalline structure

An ordered arrangement of atoms or molecules in a solid. This ordered arrangement consists of repeating patterns of molecules.

Distilled water

Water without any impurities. Impurities are removed from the water through a process known as distillation.

Latent energy

Refers to the energy released or absorbed by a thermodynamic system at a constant temperature.

Seed crystal

An initial crystal that allows for the continued process of crystallization.

Supercooled liquid

A liquid with a temperature below its freezing point but which is still in the liquid state.

Step 1 Fill two plastic water bottles with distilled water and refrigerate them overnight. Although only two water bottles are used in the experiment, placing extra bottles of water in the fridge is advisable so that the experiment can be attempted multiple times.

Fill the container with ice.

Submerge two refrigerated plastic water bottles in the ice, making sure all surfaces of the bottles are in contact with the ice.

Generously sprinkle rock salt all over the ice.

Insert a thermometer between the bottle and the ice in order to monitor the temperature of the bottle.

Carefully monitor the temperature of the bottles in the container until it drops to -8oC. If the temperature falls below -8oC, the water can freeze prematurely. During this period of observation, add extra ice and salt to the container as needed to keep the bottles submerged in the ice and salt mixture.

After the water has been at -8oC for 10 minutes, remove the bottle from the container, and note that it is still in liquid state. Strike the bottle sharply against a table and observe what happens.

Try pouring the supercooled water onto an ice-cube and see how high an ice tower you can create.

The bottles used should be made of plastic, not glass. Striking a glass bottle on a table may cause it to break and produce a health hazard.

You and a friend are hanging out on a hot day and you decide to play a prank on them. Offering your friend a bottle of water you reach into an ice bath and pull out a strangely cold bottle of water. As you are about to hand it to them you ask them if they wanted ice with their water, then strike the bottle against a hard surface. The water seems to instantly freeze right before your eye. “How did you do that?” Your friend asks, amazed!

A little bit of advice:

It is very difficult to get this experiment to work correctly the first time around and that is why this demonstration is not ideal for festivals. This experiment is impressive, but it is highly sensitive to small changes in variables, so the result can be very much hit-or-miss. To increase the likelihood of success, during the demonstration, document the amounts of ice and salt used, and take measurements of the time that bottles are submerged in the ice and salt. This can help you to keep track of the experimental conditions and if the experiment is a failure, these recordings can help you avoid repeating the mistake by changing some experimental variables, for example by increasing the amount of salt used or the time of submersion.

For how long does the water need to be supercooled? If the water is already refrigerated, it should be kept in ice for at least 10 minutes.

Why is it so difficult to get the experiment right the first time around? The success of the experiment depends on many variables; such as temperature, and the proportions of ice and salt. Small changes in these variables can lead to success or failure.

Does it only work with pure water? No, but other factors need to be considered in that case, for example the water may need to be cooled to a higher or lower temperature.

Why is the salt added to the ice? To decrease the temperature of the ice below 0oC.

Why use distilled water? Distilled water has a freezing point of 0oC. Water that is not distilled may have impurities that increase or decrease the freezing point of the water.

When water is cooled and starts to freeze, the molecules in the water come together to form a crystalline structure. The crystalline structure is what gives ice its rigidness. The water molecules in the crystalline structure have less energy than water molecules in the liquid state. This is due to the fact that in the transition from liquid to solid, energy is released as heat. This also explains why the supercooled water turns into a slush rather than a solid chunk when it freezes instantly. The heat released from the instant formation of ice prevents the formation of a solid ice block.

For ice to form as the water cools, the initial ice crystals need an object on which to grow around. In normal water, ice usually grows on microscopic impurities found in the water, however, if distilled (or very pure) water is used, it contains no such impurities. Thus, the water can be cooled below its freezing point of 0oC without actually freezing and remaining as a liquid, in a process known as supercooling. Striking the bottle against a hard surface (such as the table) makes it more likely that some of the molecules in the water move together to form a crystal structure. Once a few of the molecules join together, the crystal can grow around them. Thus, the molecules quickly join together, and the crystal structure spreads throughout, causing the water in the bottle to freeze almost instantly. ( https://www.stevespanglerscience.com/lab/experiments/instant-freeze-soda-ice/ )

The theoretical freezing point of water is 0oC, when water is cooled below this temperature, it can be expected to change state from a liquid to a solid, ice. However, in this experiment the distilled water was supercooled: its temperature was lowered below 0oC, but it remained a liquid until it was struck on a hard surface.

Heat energy plays a key role in changes of state. In the transition from solid to liquid, for example from ice to water, heat energy needs to be provided in order to break the crystal lattice in the ice. In order for a liquid to transition to a solid, it must lose heat energy through the cooling process. However, simply cooling a liquid is not enough for water molecules to arrange themselves into solid crystals, extra energy must be provided to initiate the process of crystallisation, known as the latent heat of fusion. As soon as this energy has been provided and a small crystal is formed in the liquid, the rest of the molecules in the liquid quickly rearrange themselves into a crystal structure and the water freezes to a solid. The small crystal which triggers the crystallization process is called a seed crystal. In the case of this experiment, the latent heat of fusion is provided by striking the bottle against a table. This latent heat energy is enough for the water molecules to group and form at least one seed crystal.

Salt was mixed was added into the container of ice, to help supercool the water. On its own, ice would have maintained a temperature in the region of 0oC, which would have been too high to supercool the water to -8oC. Adding salt to the water had the effect of lowering the freezing point of the ice and allowing it to maintain a cooler temperature, making it appropriate for use in supercooling. The process of lowering the freezing point of a substance is known as freezing point depression.

Applications

Heat packs are used to warm parts of the body in order to relieve pain. Some heat packs use chemical reactions that release thermal energy i.e. exothermic reaction. In some packs, supersaturated sodium acetate is heated until it melts into a liquid. As it cools it remains as a liquid until a nucleation site is created, and then the sudden crystallization of the sodium acetate releases thermal energy. ( http://materiability.com/portfolio/latent-heat-storage/ )

The concept of latent is being implemented in novel ways to improve the efficiency of heat pumps. https://cordis.europa.eu/project/rcn/15596_en.html

Experiment with different types of water (for example tap water or flavoured water) and observe the behaviour of the water as it freezes. https://www.stevespanglerscience.com/lab/experiments/instant-freeze-soda-ice/

Investigate the effects of cooling the distilled water to different temperatures below 0oC.

Preparation: At least 1 day

Conducting: 5 mins

Clean Up: 15 mins

Number of People

1 participant

Crushed salt Ice Large container Rock salt Several bottles of purified or distilled water Thermometer

Contributors

Additional Content

Instant Freeze Water – Bottle Slam (Beginner)

Measuring the Latent Heat of Fusion of Ice  (Intermediate)

Phase Change and Latent Heat  (Advanced)

Cite this Experiment

Vella, R., Padfield, N., & Styles, C. (2020, August 25). Instant-Freeze Water. Retrieved from http://steamexperiments.com/experiment/instant-freeze-water/

First published: August 25, 2020 Last modified: August 25, 2020

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Unveiling the Magic of Supercooling: How to Instantly Freeze Water

Table of contents.

Have you ever marveled at a magic trick where water turns into ice instantly? What if I told you it’s not magic but science at its best, and you can recreate this phenomenon at home? Welcome to the fascinating world of supercooling, a process that defies our everyday experience with freezing and offers a peek into the intriguing behaviors of liquids under extreme conditions.

What is Supercooling?

Supercooling is an extraordinary state where a liquid or gas is cooled below its usual freezing point without it transitioning into a solid form. For water, this means having liquid water exist below 0 degrees Celsius (32 degrees Fahrenheit) without it turning into ice. This phenomenon challenges our conventional understanding of freezing and provides an exceptional opportunity to explore the delicate balance between temperature and state of matter.

The Science Behind Supercooling

At its core, supercooling is all about achieving a state of delicate equilibrium. Under normal circumstances, when water reaches its freezing point, it begins to form ice as the molecules slow down and arrange themselves in a crystalline structure, releasing energy in the process. However, if the cooling process is smooth enough to avoid disturbing this delicate balance, water can be cooled below its freezing point without the formation of ice crystals. This state is unstable, though; even a minor disturbance can trigger rapid freezing.

The Instant Ice Phenomenon

The most mesmerizing aspect of supercooling is what happens when this unstable state is disturbed. Imagine a bottle of supercooled water, liquid and clear, sitting well below freezing temperature. The moment you pour it out or introduce an impurity, ice forms instantly right before your eyes, as if by magic. The transition from liquid to solid happens so quickly because the supercooled water was just waiting for an excuse to freeze. During this instant freezing process, the water actually warms up to 0 degrees Celsius as it releases latent heat.

Experimenting with Supercooling at Home

Curious to try this at home? With patience and careful preparation, you can witness the magic of supercooling in your own kitchen. Here’s how:

Materials You’ll Need:

• Purified or distilled water (Impurities in regular tap water can initiate freezing)
• A clean and smooth plastic bottle (Avoid scratches that can act as nucleation sites for ice formation)

Step-by-Step Guide:

• Prepare Your Water: Fill the plastic bottle with purified or distilled water. Make sure the bottle is smooth and free of any labels or residues.
• Chill: Place the bottle in your freezer. The key here is to find the sweet spot where the water is supercooled but not frozen. This usually takes between 2 to 3 hours, but it can vary depending on your freezer’s temperature settings.
• Check Carefully: After a couple of hours, gently check the bottle every 15-20 minutes. You’re looking for water that’s still liquid but is very cold.
• Initiate Freezing: Once you have your supercooled water, remove it from the freezer carefully to avoid disturbing it too much. For the grand reveal, either pour the water onto a piece of ice or tap the bottle gently. You’ll witness the instant transformation from liquid to ice.

Safety Tips:

While experimenting with supercooling is generally safe, always handle bottles with care as they can crack under extreme temperatures.

Understanding Supercooling’s Implications

Beyond being a captivating demonstration, supercooling has profound implications in various scientific and practical fields. In meteorology, understanding supercooled water droplets helps in predicting weather patterns and phenomena like freezing rain. In technology, researchers are exploring ways to use supercooling in preserving organs for transplants more efficiently than current methods allow.

The Wonders of Water

Water continues to surprise and intrigue scientists with its unique properties and behaviors like supercooling. It’s a reminder of how even the most common substance on our planet has secrets waiting to be unlocked. Supercooling exemplifies how science can turn everyday materials into objects of wonder and exploration, bridging the gap between the laboratory and your kitchen.

So next time you see a magic trick where liquid instantly turns to ice, remember: it’s not just an illusion but a delightful demonstration of physics at play. And with a bit of patience and precision, it’s something you can experience firsthand from the comfort of your home.

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Taking Apostolic Children's Ministry to the Next Level

How to Instantly Freeze Water

by James Wang Leave a Comment

This Science experiment will teach your Sunday School class how to instantly freeze water. To do this, you just need a purified bottled water, a glass and a small piece of ice.

Though it is the easiest Science experiment you will ever do with the kids, what you and your class would need is patience. In order to accomplish, you need to let the purified bottled water sit in the freezer for two hours. This will allow the water to hit below its natural freezing point. Once it is super chilled, pour it in the glass and drop the piece of ice. The reaction is super amazing as the water will freeze right before your eyes like magic.

For more amazing Science experiments, visit One Crazy House .

Making Water Freeze Instantly – Supercooling a Bottle of Water

What happens when you leave a bottle of pure water in the freezer for a few hours? Weirdly it doesn’t freeze until you bang the bottle. Why?

This is special bottled water. It’s not regular mineral water, which normally has other chemicals dissolved in it. Smart Water claims to be pure distilled water, or near enough.

After a few hours in the freezer, the water should be around -20C but it will still be a liquid. Why?

Regular water, at 0C will begin to freeze. To form the ice needs a “seed” – a speck of dust or another chemical impurity that provides a site for the first ice crystals to begin. This is called nucleation .

Because it’s so pure, it’s possible for the smart water to exist in a supercooled state . There is nothing in the water to kick start the nucleation process off.

If I then bang the bottle, give it a shock, the shockwave provides the kick start to begin the freezing process.

In theory, it should be possible to cool the water down to -48 degrees Celsius before a different process kicks in and freezing will happen anyway.

Try it for yourself. Get a bottle of Smart Water , put it in the freezer for a few hours, then give it a shock. The best bit is you can then let it thaw out fully, then try it again!

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Science News Explores

Build ice towers with bottled water and ice.

Add cold water and salt to create the conditions for your own mini ice castle

Bottles of water can be poured to create a tower of solid ice if the water is cold enough.

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By Bethany Brookshire

September 16, 2020 at 6:30 am

This article is one of a series of  Experiments  meant to teach students about how science is done, from generating a hypothesis and designing an experiment to analyzing the results with statistics. You can repeat the steps here and compare your results — or use this as inspiration to design your own experiment.

In the Disney movies Frozen and Frozen II , Elsa uses magical powers to make castles of ice. We can’t build castles, but we can pour out small ice towers. Elsa may have magic. We use science. 

In an earlier experiment, I showed that sugar seed crystals are important for making rock candy. Those crystals provide a nucleation point where the sugar structure — the candy — can self-assemble. But sugar isn’t the only substance that can form a structure with the right nucleation point. Water can, too. It just needs to be super cool.

If you pour extremely cold water (water that’s less than 0° Celsius or 32° Fahrenheit) onto an ice cube, that cube acts as a nucleation point. The water will freeze when it hits the cube, and then stack up — forming a tower of ice.

If the water isn’t cold enough, though, you just get a lonely ice cube in a puddle of water.

As water freezes at 0 °C, I need a way to chill water to cooler than that. And I can’t let it freeze until it comes into contact with the cube. How? The secret is salt.

Saltwater has a lower freezing point than freshwater. This is why you can spread salt on an icy sidewalk and the ice will melt. Salt crystals in the water prevent the water molecules from linking up to form a crystal. Saltwater needs to be even colder than freshwater to freeze — cold enough that the salt gets pushed out of the water’s crystal structure. Freshwater freezes at 0 °C (32 °F), but saltwater freezes at –2 °C (28.4 °F).  

Is salt the difference between an ice tower and a puddle? I’m going to do an experiment to find out.

I need to start with a hypothesis — a statement I can test. Here, my hypothesis is: Water chilled in ice with salt will be colder and form taller ice towers than water chilled in water without salt .

To test this, I start with two small coolers. One cooler just has ice. The second cooler has ice mixed into a slush with salt.

I place bottles of purified water into each cooler. Be careful to check the label when you buy the water. The water should be purified or distilled. It should contain no added minerals. Those minerals might serve as nucleation points — making the bottled water freeze into a solid block of ice as soon as it gets cold enough to freeze. But if the water is pure, it should be able to get colder than 0 °C (32 °F) and stay a liquid.

Just one bottle of water in each cooler isn’t enough. I want to make sure that I can collect enough data to detect a difference between water chilled in fresh or salty ice. So I put eight bottles of water into each cooler.

Here’s how how to build ice towers:

• Fill two smaller coolers with ice (or you can use one cooler, and run the experiment twice).
• In one cooler, add 700 grams of salt (24.7 ounces, or about 2.5 cups). With a spoon, mix the salt and ice together very well. The ice will immediately start melting into a thick slush.
• Sink eight bottles of purified water into each cooler (for a total of 16). Make sure the bottles are each evenly surrounded with ice or slush.
• Wait 25 minutes.
• Gently rotate the bottles in the ice or slush. Make sure you do not shake the bottles.
• Wait another 20 minutes.
• Take the temperature of the bottles. I did this with a no-contact thermometer, but you can also use a regular thermometer; sink it into the ice next to the bottles.
• Place an ice cube on a plate. Using a ruler, measure the height of the cube.
• Remove a bottle of water carefully from the ice or slush and open it. Again, be careful not to shake it. (If you shake the super-cooled water, air inside the bottle will mix with the water. That creates nucleation points — and your bottled water will freeze before you open it.)
• Gently and slowly pour a small, steady stream of water onto the ice cube. After a second or two, the water will begin to freeze on top of the cube, producing an ice tower.
• Quickly whip out your ruler and get the height of your ice tower before it melts. (A friend is helpful for this.)
• Repeat until you’ve tested all 16 of your bottles.

Results on ice

To calculate how much ice I was able to grow, I subtracted the height of the ice cube from the height of the final ice tower. I added that to a spreadsheet with the temperature of each ice bottle.

The bottles chilled in ice got pretty cold — an average of 1.16 °C (34.1 °F). But the bottles chilled in salt slush got colder — an average of –2.65 °C (27.2 °F).

To confirm that these two groups are indeed different, I need to run statistics . These are tests to interpret the meaning of my results. I ran a t test. This test finds differences between two groups. Lots of websites online will let you plug your data in to run these tests for free. I used one from GraphPad Prism .

The test gave me a p value. It’s a measure of how likely it is that I would by accident find a difference as big as the one I found here. Many scientists consider a p value of less than five percent (0.05) to be statistically significant. This means that I would have a five percent chance of finding a difference that size by accident alone. If the p value I get is less than 0.05, I have a statistically significant difference.

For the two temperatures in my experiment, the p value was 0.0001, or 0.01 percent — definitely significant.

But just because there is a difference doesn’t mean that this difference is large. Tiny differences can be statistically significant. So I also ran a Cohen’s d test. Here I had to calculate my standard deviation. This is the amount that each set of data differs from the mean (or average). You can calculate that in any spreadsheet on a computer. On mine, I used Microsoft Excel, and used the function “= STDEV” and highlighted my data set. Then I put my mean, standard deviation and number of samples into this online calculator .

My Cohen’s d result was 2.93. Many scientists consider a Cohen’s d higher than 0.8 as a large effect size. So, the water cooled in the salt slush was definitely colder than the water cooled in simple ice.

As for the towers I built, the difference looked pretty obvious. When I poured out the bottles chilled in clean ice, I just got a puddle of water around my ice cube. Not a single one grew a tower. The difference between the ice cube and the final tower height was zero every time. But my saltwater-chilled bottles produced visible towers. Their average height was 5.66 centimeters (2.22 inches).

I ran statistics for these results as well. The p value for my final towers was 0.0001. My Cohen’s d was 4.13. Salt water made a very big difference in tower height.

My hypothesis when I started was that water chilled in ice with salt will be colder and form taller ice towers than water chilled in water without salt . Based on my results, it seems I was right. Salt helped water get colder and grow taller towers.

In every experiment, there are always things I could have done differently. In this one, I ended up with only six usable saltwater-chilled water bottles. The other two froze. Water in those bottles may not have been quite as “pure” as advertised on the label. I would probably chill extra bottles next time. Not only that, I didn’t always pour out the exact same quantity of water. Next time, I could weigh my results, and make sure that I poured out the same mass of water each time. That might help me get even more accurate results.

Luckily, this experiment is cheap. And I can easily run it again.

• Ice (two bags): $1.99 each
• Purified water (two packs of eight): $3.28 each
• 9-quart cooler (two): $9.97 each
• Salt (three pound box): $2.63
• Handheld infrared thermometer: $18.00
• Ruler: $0.97

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How to Supercool Water

November 17

• Acquire distilled or purified water.
• Fill an empty bottle with tap water (this is your control).
• Place all three bottles in the freezer at the same time
• Leave them in for roughly 2 hours (if all of the water was originally at room temperature)
• The timing of this will vary with each freezer. After the first hour, check on the bottles periodically to check for signs of freezing.
• The bottle filled will freeze before the purified water. At this point you will know that the purified water is below zero, and is ready to be removed from the freezer.
• Take it out, give it a hard slam on the table, and watch the H 2 O turn from liquid to a solid right in front of your eyes!

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How to Freeze Water Fast

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You won’t believe your eyes when you watch a glass of water instantly freeze when an ice cube is placed in the water! Let’s get started with this super cool science experiment and learn How to Freeze Water Fast.

Get more fun and educational Weather Experiments for Kids here!

This is such a fun and fascinating experiment and a great way to teach kids about the freezing point of water!

Table of Contents

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Supplies Needed:

• 2 or 3 Sealed Water Bottles
• Thermometer
• Clear Glass Cup

How to Freeze Water Without a Freezer

• Fill a large bowl with ice, 2 or 3 sealed water bottles, and some rock salt.
• Stick a thermometer into the bowl of ice and wait for the temperature of the ice water in the bowl to drop to 17º-20º F.
• Gently pull one of the bottles out of the bowl and pour it into a clear glass cup or jar.
• Grab an ice cube and place it in the glass of very cold water to watch the water instantly freeze into ice!
• Repeat steps 3-4 with your remaining supercooled water bottles to watch the ice form before your eyes again and again!

Step 1: Add Ice, Rock Salt, and Water Bottles Into a Large Bowl

Although this super cool experiment does freeze water in an instant, there is a little bit of prep time that we need to do first to get the temperature of the water bottles to just the right point where they will freeze!

So go ahead and fill a very large bowl with lots and lots of ice cubes! Then stick 2 or 3 sealed water bottles into the icy bowl.

Now scatter some rock salt on top of the ice in the bowl. Be very generous with the rock salt. The properties of salt actually allows ice to melt at a temperature below the freezing mark (32°F or 0°C).

As the ice melts into an icy-watery mixture in the bowl, it will remain below freezing thanks to the salt. This will cause the water inside the water bottles to drop below freezing without turning to ice!

Step 2: Use a Thermometer to Monitor the Temperature of the Ice

Grab a cooking, or meat thermometer and place it in the ice near the bottles. It should take at least 30 minutes for the temperature to get where it needs to be, but could take up to 90 minutes.

This step is probably the hardest part if you lack patience like I do…but it’s crucial in order to get the water in the bottles to be supercooled to the right temperature.

Supercooling the water is the scientific term for when a temperature of a liquid drops below it’s freezing point and remains a liquid (water) without turning to a solid (ice).

We need the temperature of the ice water in the bowl to be between 17°-20°F for the water inside the bottles to get supercooled.

If you don’t have a thermometer on hand, another great sign that your water bottles are cold enough to make liquid ice is to check the outside of the bottle for frozen condensation droplets.

If the condensation on the outside of your bottles have become frosty or frozen, the liquid inside the bottle is likely cold enough to move on to the next step to make water freeze fast!

Step 3: Pull a Bottle Out and Pour it Into a Clear Glass

Once you determine that your water bottles have cooled to that magical 17°-20°F temperature range, gently pull one of them out of the bowl.

Just a slight shake of the bottle could be enough to prematurely kickstart the freezing process and you will end up with a frozen bottle of water (which is still fascinating, but not the end result we want this time).

While being careful not to disturb the water more than you have to, pour one bottle of water into a clear glass cup or jar.

It’s best to fill the glass very close to the top with the supercooled water out of the bottle.

Depending on the size of your glass you may need to use 2 bottles, but I prefer to save the 2nd and 3rd bottles to repeat the process you are about to witness!

Step 4: Touch an Ice Cube to the Water in the Cup

With your glass nearly filled to the brim with liquid water that is below the freezing point, grab an ice cube and place it in the top of the water.

Depending on the temperature of your water, it might take a few seconds for the reaction between the ice cube and the supercooled water to be seen.

After a few seconds the water in the jar will begin to crystalize and solidify into a slushy, icy drink! It is mesmerizing to watch this ice making process unfold right before your eyes!

Step 5: Repeat the Process With Multiple Bottles to See the Ice Form Again

Now that you have learned how to freeze water fast why not have some fun making more ice with your other bottles that are supercooled!?

You can repeat this same super cool experiment again by repeating steps 3-4, or you could try some other cool things with your supercooled water like making ice pillars in this How to Turn Water Into Ice Instantly experiment!

Can Ice Cubes Freeze Water?

It is more typical that ice cubes melt into a glass of cold water than it is for ice cubes to freeze a glass of water. This is because water needs to lose a lot of heat energy to drop below freezing.

Most household ice is at a temperature of about 23°F, so it doesn’t take much heat energy from water to melt the ice cubes, however there are a few cases in which ice cubes can freeze water!

In our experiment how to make water freeze fast, the water bottles were already supercooled and remained as a liquid below freezing.

The below freezing water just needed an ice cube or particle to crystalize onto to start the ice forming process.

Adding the ice cube to the supercooled water did turn the water into ice because the water was already below freezing.

Another way to turn water into ice with an ice cube would be to use ice cubes that are very cold to allow them to absorb enough heat energy from the water without the ice cubes melting.

This typically doesn’t happen unless the environment (outside, freezer, etc.) is below freezing and freezes the water and keeps the ice frozen too!

How Does Water Freeze Into Ice?

Liquid water typically freezes into a solid form of ice when the temperature of the water drops at or below 32°F. This is the temperature that water molecules slow down enough to stick to each other and form a solid crystal.

From this experiment, we learned that water does not always follow the rule of freezing at 32°F though. Under the right conditions, purified water that does not have impurities and minerals can drop below the freezing point and remain a liquid and is referred to as supercooled water.

What causes freezing rain?

Freezing rain is a great example of supercooled water in the atmosphere. This fascinating weather phenomenon occurs when warm air (above freezing) rises up and over cold, (below freezing) air at the surface.

As the precipitation falls out of the clouds into the warmer air, it melts and becomes all rain until it reaches the thin layer of cold air near the ground. This cold layer of below freezing air near the ground supercools the rain drops.

Even though the raindrops have been supercooled to a temperature below freezing, they do not have time to freeze during their short trip through the shallow layer of cold air.

These below freezing supercooled raindrops then freeze immediately on contact with anything they contact at the surface including cars, roads, trees, sidewalks, etc. Freezing rain storms are often called ice storms and can make very dangerous winter conditions.

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More Fun and Educational Experiments for Kids:

• How to Make Instant Snow at Home
• Flexible Eggshell Experiment
• Baking Soda and Vinegar Balloon Experiment

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• Experiments / Science

How to freeze a glass of water instantly

by Scott Dutfield · 13/05/2021

Get the power of a superhero and freeze a glass of water with a single touch!

If you’re under 18, make sure you have an adult with you.

1. Cool your water

First, you need to freeze some purified water. You might think that you can make your own purified water for this experiment by boiling it for a few minutes, but that won’t remove the chemicals in the water, so you’ll need to buy specially purified water instead. Take three unopened 500ml plastic bottles of the water and place two of them in the freezer on their side.

2. Be careful!

After 30 minutes put in a third bottle. Having more than one bottle will increase your chances of this working, so you can put in even more if you want to try this a few times! You need to leave your water in the freezer for two hours and 15 minutes in total. Make sure to leave the water as undisturbed as possible while it’s in your freezer, as agitation can start the crystallisation process.

3. Carefully remove it

After two hours and 15 minutes, slowly open the freezer and very carefully remove the lid of the bottle. If the process has worked correctly the water should still be liquid, but it will have been supercooled to below its freezing point. Tilt the glass you’re going to use and slowly pour the water into the glass. If you’re careful, the supercooled liquid shouldn’t start to solidify.

4. Grab some ice

You’ll need some crushed ice for this part. Put your finger into the crushed ice and make sure that there’s at least one ice crystal stuck to your fingertip. That’s all it will take to start the crystallisation process in the rest of the water. When you’ve got a crystal on your finger, gently lower your finger into the glass of supercooled water and watch what happens.

5. How did that happen?

If everything has worked properly the water should instantly start to solidify, with ice crystals spreading through the water to make ice. If you want to skip this step you can always just leave the water inside its plastic bottle and hit it on the side to kick-start the process. That one small movement is all that’s needed to start a chain reaction through all the molecules in the water!

In Summary…

Tap water will usually freeze at 0°C because of the chemicals and impurities in the water. The molecules can latch onto these impurities, and freezing is simple. In purified water there are no impurities, so if you’re careful the water can be cooled to well below its normal freezing point.

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How to Supercool Water

Last Updated: July 16, 2024 References

This article was co-authored by Meredith Juncker, PhD . Meredith Juncker is a PhD candidate in Biochemistry and Molecular Biology at Louisiana State University Health Sciences Center. Her studies are focused on proteins and neurodegenerative diseases. There are 8 references cited in this article, which can be found at the bottom of the page. This article has been viewed 380,605 times.

Supercooling a liquid is when you cool it to below its freezing point without it becoming a solid. [1] X Research source Supercooling only works with liquids that have no impurities that can trigger crystallization. Once the liquid is supercooled, you can trigger it to form ice right in front of your eyes. Although it may sound complicated, you can supercool water in your very own home with a few simple steps.

Using a Bowl of Ice and Salt

• Make sure your glass is very clean; any impurities can cause the water to crystallize into ice before supercooling occurs. Impurities can also lower the freezing point of water. [2] X Research source
• The bowl must be large enough to contain the glass and enough ice to submerge the glass.

• Be careful not to drop any ice into the cup of water.
• Covering the cup before you add ice and salt is a good way to prevent accidental contamination.
• Again, be careful not to get any salt into the glass/cup.
• At this point you can add a clean thermometer if you have one.
• This process generally takes about 15 minutes, plus or minus a few minutes depending on your personal freezer settings. If you leave the water for too long, it will freeze.
• If you don’t have a thermometer, you can set-up a second bowl with tap water. When the tap water freezes, your purified water should be super-cooled.

Using the Freezer

• Water expands as it freezes, so ensure the bottle isn’t completely full before you proceed.

• If you live where the temperature outside is below freezing, you can put the bottle of water outside.

• Starting at 2 hours, check the bottle of tap water every 15 minutes to see when it freezes.
• When the tap water is completely frozen, the pure water will be supercooled.
• If your pure water is also frozen, you may have waited too long, bumped the bottle during the process, or the water wasn’t completely pure.
• Melt the water and try again for a shorter amount of time.

Expert Q&A

• Supercooled water is still drinkable and you can also make a slushie out of the slush. Thanks Helpful 12 Not Helpful 10

• Don't let it stay in there too long! Thanks Helpful 44 Not Helpful 11
• When water freezes it expands, so make sure that the bottle isn't completely full when you put it in the freezer. Thanks Helpful 49 Not Helpful 16

Things You'll Need

• Distilled water
• Two water bottles

You Might Also Like

• ↑ https://www.esrf.fr/news/general-old/general-2010/supercooling;jsessionid=5141EA5A0B183F530F1DE4D5D034AA79
• ↑ https://astrocampschool.org/cool-water/
• ↑ http://www.sciencebuddies.org/science-fair-projects/project_ideas/Phys_p033.shtml#procedure
• ↑ http://physicsbuzz.physicscentral.com/2014/06/the-science-of-ice-cream.html
• ↑ http://www.sciencebuddies.org/science-fair-projects/project_ideas/Phys_p033.shtml#background
• ↑ http://chemistry.about.com/od/chemistryhowtoguide/a/how-to-supercool-water.htm
• Videos provided by HouseholdHacker

About This Article

If you want to supercool water using salt and ice, fill a clean glass 1/4 of the way full of purified water, then place the glass in the center of a large bowl. Add enough ice to the bowl so the glass is completely surrounded, then sprinkle 2 tablespoons of salt over the ice. Take care not to get any ice or salt inside the glass. Wait about 15 minutes, or until a thermometer shows that the water is below 0°F. To crystallize the water, either drop a piece of ice into the glass or pour the water onto ice. You should see ice form almost immediately. To learn tips from our reviewer on how to supercool water in the freezer, keep reading! Did this summary help you? Yes No

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Supercooling Water – 2 Easy Ways to Supercool Water

Supercooling water is easy and fun. Basically, you chill water below its freezing point and crystallize into ice on command. Here are step-by-step instructions for supercooling water using two different methods.

Method #1: Supercooling Bottled Water

Supercooling bottled water in a home freezer is the easy. The only “trick” is that the bottled water needs to be reasonably pure. Fiji water is one of the most reliable brands to use, but any water purified by reverse osmosis or distillation works well. The reason bottled water works better than tap water is because tap water contains tiny particulates, dissolved minerals, and dissolved gases that provide nucleation sites for ice to form. You can still supercool tap water, but first let it sit for several hours so any bubbles can escape.

• Place an unopened bottle of water in the freezer. Also, fill a bottle the same size with tap water.
• Let the bottles chill undisturbed for around 2-1/2 hours. The exact time needed to supercool water depends on the temperature of the freezer and the size of the water bottle. Don’t disturb either bottle, but look to see if the tap water has frozen. When the tap water freezes, it means the purified bottle water is supercooled. (If the water in both bottles is frozen, you need to melt the ice and start again, but check on the bottles after less time. If both bottles freeze again, either you waited too long again or else the bottled water wasn’t sufficiently pure.)
• Carefully remove the bottle of supercooled water from the freezer.
• Initiate crystallization into ice by shaking the bottle or by opening it and pouring the liquid over a piece of ice or bit of frozen popsicle.

Method #2: Supercool a Glass of Water

You don’t need a freezer to supercool water. All you need is ice and some salt. When you sprinkle salt on ice, the small amount of melted ice (liquid water) dissolves salt and experiences freezing point depression. This lowers the temperature of the ice below 0 °C or 32 °F. It’s a handy way to freeze homemade ice cream that also works for supercooling water.

• Fill a large bowl with ice. Sprinkle a couple of tablespoon of salt over the ice.
• Either chill a water bottle (with bottle of tap water, for reference) in the salted ice or place a glass of purified water in the bowl (with thermometer, so you know when it’s supercooled). Make sure the level of the ice is above the liquid level in the container.
• For a glass containing only a couple of tablespoons of water, it only takes around 15 minutes to supercool the liquid. Bottled water takes longer and you may need to refill the ice in the bowl if too much melts.
• Freeze the supercooled water into ice by disturbing it or by dropping a small piece of ice into the water.

How Supercooling Water Works

Water normally freezes into ice at 0 °C or 32 °F, but it can be supercooled at ordinary pressures down to a temperature as low as −48.3 °C or −55 °F, as long as there are no nucleation sites to support ice crystal growth. At the bottom end of the temperature range, supercooled water is glassy water. In other words, it is an amorphous solid, but not crystalline ice. But, just below the freezing point it’s still a liquid.

As temperatures drop below freezing, supercooled water doesn’t crystallize because of the way water molecules organize when chilled. Normal ice is hexagonal ice (like six-sided snow crystals), but hydrogen bonding gives water other options. Supercooled water has tetrahedral water molecules and pentameric water clusters. So, even though hexagonal ice is more stable than liquid water, it doesn’t form its preferred crystal structure unless it is disturbed because the tetrahedral and pentameric water is “in the way.”

Supercooling vs Freezing Point Depression

Some people confuse supercooling with freezing point depression . While both processes cause a liquid to freeze at a temperature below the normal freezing point, supercooling involves a pure substance. Freezing point depression occurs when dissolved particles interrupt the organization of molecules from the liquid to solid phase.

Examples of Supercooled Water in Nature

Supercooling of water is a natural phenomenon. It occurs in cumulus and stratus clouds. Aircraft employ de-icing systems because otherwise supercooled water crystallizes into ice when the wings disturb it.

Freezing rain is another example of supercooled water. When the rain strikes a surface, it crystallizes into ice.

If you enjoyed this project, another fun example of supercooling is hot ice !

• Chaplin, Martin (2004) “ Supercooled water .” Water Structure and Science.
• Debenedetti, P. G.; Stanley, H. E. (2003). “Supercooled and Glassy Water.” Physics Today . 56 (6): 40–46. doi: 10.1063/1.1595053
• Giovambattista, N.; Angell, C. A.; Sciortino, F.; Stanley, H. E. (July 2004). “Glass-Transition Temperature of Water: A Simulation Study.” Physical Review Letters . 93 (4): 047801. doi: 10.1103/PhysRevLett.93.047801
• Moore, Emily; Molinero, Valeria (2011). “Structural transformation in supercooled water controls the crystallization rate of ice.” Nature . 479 (7374): 506–508. doi: 10.1038/nature10586

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Follow-up #2: supercooled water.

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Follow-up #9: more data, follow-up #10: more ice data.

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Follow-Up #11: gas in soda

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Follow-Up #12: Navy weighs in

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Follow-Up #15: supercooling under pressure

I'd be surprised if you found a sharp pressure threshold for super cooling. Usually even tap water will supercool a bit under atmospheric pressure. If you do find that the amount of supercooling you can get does depend a lot on pressure, here's a possible reason. Maybe the main nuclei are little bubbles. If you pressurize the water, you can shrink or eliminate them. Then you'll just be left with dust, etc. for nuclei. So you might find that the amount of supercooling you can get drops for the first bit of pressurization, then levels off for further pressurization. The reason that atmospheric pressure would be special is that the air dissolved in the water has had a chance to reach equilibrium with the atmosphere, so increasing pressure leads little bubbles to dissolve. Since you find that taking the pressure off usually leads to freezing, I guess the bubbles have shrunk under pressure but not completely gone away. Maybe the one time that releasing the pressure didn't cause freezing, you'd actually managed to fully remove the bubbles. Slightly depressurizing the water for a while, then letting it come back to atmospheric pressure without shaking it should remove bubbles. That too should increase supercooling. You can see I'm doing a lot of guessing, but these are all testable ideas. One tool that might help would be a laser pointer. In a dark room, you can see how much the water scatters light. That scattering mostly comes from bubbles, dust, etc. Mike W.

(published on 05/07/2011)

Follow-Up #16: supercooled slush and latent heat

Great observation and great question. When the freezing starts, the water molecules joining the ice lose energy. That's the whole reason they join the ice, the bonds between them in the ice are lower energy than the looser bonds in the liquid. That lost intermolecular bond energy (called latent heat) has to go somewhere. It heads straight into the vibrations of the nearby molecules, heating them up. That keeps happening until the water is back up to 0°C, at which point the freezing stops. We've discussed this a little on some previous answers. The latent heat is big enough that for practical supercooling it will stop the freezing well before 100% of the water has frozen. That's why you end up with a slush. (For purists, yes we should be talking about enthalpy rather than energy, but there's not much difference here.) Mike W.

(published on 05/10/2011)

Follow-Up #17: supercool slurpies

(published on 07/06/2011)

Follow-Up #18: thumping supercooled water

(published on 07/12/2011)

Follow-Up #19: Supercooled water project

(published on 09/12/2011)

Follow-Up #20: pesticide impurities in slushy ice-water?

(published on 11/08/2011)

Follow-Up #21: possible supercooling?

(published on 11/09/2011)

Follow-Up #22: more supercooling data

(published on 12/28/2011)

Follow-Up #23: our staff

(published on 03/16/2012)

Follow-Up #24: supercooled water cooler

(published on 03/31/2012)

Follow-Up #25: supercooled margaritas

(published on 06/03/2012)

Follow-Up #26: nucleating supercooled water

(published on 06/11/2012)

Follow-Up #27: surprise supercooling

(published on 07/01/2012)

Follow-Up #28: Thank you for the thanks

(published on 08/09/2012)

Follow-Up #29: slush from supercooled water

(published on 11/07/2012)

Follow-Up #30: supercooled water viscosity

(published on 12/14/2012)

Follow-Up #31: freezing supercooled foam!

(published on 12/26/2012)

Follow-Up #32: supercool experiments

(published on 01/03/2013)

Follow-Up #33: shaking triggers ice formation

(published on 01/20/2013)

Follow-Up #34: supercooled water and slush?

(published on 01/24/2013)

Follow-Up #35: solid ice from supercooled water?

(published on 04/08/2013)

Follow-Up #36: frozen supercooled water

(published on 04/09/2013)

Follow-Up #37: supercooling in Estes Park

(published on 04/23/2013)

Follow-Up #38: experience with supercooled water

(published on 04/28/2013)

Follow-Up #39: supercooled beer

(published on 04/29/2013)

Follow-Up #40: ice water turning to slush

(published on 05/12/2013)

Follow-Up #41: popsicle making

I'm not sure what the procedure is. Adding any solute (e.g. sugar) to water will lower its true freezing point below 0°C. I think you're talking about trying to actually supercool it, to keep it from forming big ice crystals even when it's cold enough to start freezing despite the sugar. One possible way to promote supercooling is by adding some of the special antifreeze that antarctic fish have in their blood. It's a sort of very small protein. () The concentration is too low to lower the true freezing point much but it binds to ice crystals as they just start to form, greatly slowing the formation of big crystals. That's how the fish can make it through the winter below the true freezing point of their blood. Some ice creams and popsicles are made using these materials, since they help keep big crystals from forming.

(published on 07/24/2013)

Follow-Up #42: supercooled water science projects

This won't be the easiest sort of science fair project, because it takes some care to make the supercooled water reproducibly. Still, there are several interesting questions that could be explored.

One, mentioned above, would to see what sorts of things are best at triggering the freezing. How cold does the water have to be for say a little piece of some rock to trigger the freezing? How cold for some reproducible little tap on the water to do it? and so forth. You could even try something where the size of the disturbance could be varied in a controllable way, maybe via little taps from different  size pendulums. How does the maximum temperature at which the tap works vary with the size of the tap? 

It's probably beyond the scope of what your daughter could do for the science fair, but pursuing this type of experiment far enough could lead to something systematic on the whole topic of how nucleation of the ordered phase occurs.  

Which reminds me that I've got to quit answering these and get back to helping with a research paper on that very topic, although in a different system.

(published on 09/29/2013)

Follow-Up #43: supercooled spring water?

Any water can supercool a little bit, but your guess that the unfiltered spring water may not supercool much sounds reasonable to me. Still, the label might not be right or that spring may naturally not have many good nucleation particles in it, so the only real way to tell is by experiment.

Mike W. 

(published on 10/18/2013)

Follow-Up #44: freezing supercooled saltwater

My first reaction is that it's hard to get highly reproducible results with supercooling, as we said in #42.

For background, let's try to calculate about how much that salt should lower the freezing point. 1/2 tsp is ~2.8 gm. The weight of a mole of NaCl is ~58 gm. So that batch had ~0.05 moles NaCl. 12 oz is ~ 1/3 liter. So you had up to ~0.15 M salt solution. That would give a freezing point depression of only around 0.5°C, not enough to make a huge difference in the net amount frozen, assuming that you've gotten the water and the sieve much more than 0.5°C below 0°C.

Yet this salt seems to have made a big difference in the freezing pattern, although maybe not in the total amount frozen. The different pattern will leave different amounts of liquid stuck to the surfaces, messing up the weight comparison, as you noticed. That small amount of salt doesn't change the viscosity of water much at all, so I think it's the different crystal pattern that accounts for the different amount of water stuck.

Why does the salt change the pattern of crystal growth so much? Here's a guess. As crystals start to form, they exclude the salt. That makes a layer of extra-salty water around them, suppressing further crystal growth. (The released latent heat of crystallization has a similar effect, but it diffuses away faster.) That promotes the formation of many little crystals rather than fewer bigger ones. That's how you get that slush.

(published on 11/30/2013)

Follow-Up #45: why does supercooling work?

I've moved your question to a thread on supercooling. You give us an excuse to explain the effect at a little more depth. The question is why would water stay a liquid even though it's cold enough so that the solid ice would be more stable than the liquid.

Let's start with the basics: why are the liquid and solid separate phases to begin with? Why don't they mush together on the scale of molecules?

The stable form of things is the one that minimizes the free energy (U-TS), a balance between lowering the energy (U) and raising the number of different possible micro-arrangements, measured by the entropy (S). At low temperature (T), lowering U is more important. So when water is cold, the molecules line up into the low-U crystal form, even though that lowers their S. At high T, S is more important, so they break loose and wander among more different arrangements. 

Imagine there is a little region with an ice crystal contacting a little region of liquid, near the freezing temperature. The molecules on the surface of the ice have lost entropy by lining up, but not lost much energy because on one side the liquid molecules aren't lined up with them. So molecules at that surface have higher free energy than ones in either the liquid or the solid. The molecules arrange to minimize the liquid/solid surface, separating the two phases. 

What happens if you start with pure liquid and cool it enough so the solid is more stable? Let's say a few molecules by accident happen to arrange in an ice-like pattern. Mostly, they're still at the ice surface. They have  higher  free energy than the liquid, not lower. So usually they will just roll back down in free energy to the liquid state. 

This sort of pure accidental arrangement takes a very long time to get an ice crystal started that's big enough to keep growing, unless the liquid is way below the freezing point. That's why you can supercool water until some special nucleation gets the freezing going.

(published on 01/01/2014)

Follow-Up #46: supercooled wine

For starters, the freezing temperature of the wine is around -6°C thanks to the alcohol and other solutes. See . So -20°C is not as much supercooled for wine as it would be for water. Still 14° or so supercooling seem large, especially for something with some sediment in it. The solute molecules themselves would not serve as nucleation centers. Other readers have written in of problems with wine freezing, so perhaps you have an unusual wine, maybe filtered. (See .)  So I'm not sure how weird your result is. 

As for the heat of fusion, it's about the same for supercooled liquids as for ones right at the freezing point. It shrinks below the true freezing point by an amount proportional to the difference between the heat capacities of the solid and liquid. That's not typically a big deal for ordinary supercooling. The latent heat of the alcohol solution, however, is a bit different from that for pure water. (see ) I doubt that difference matters much for what you're seeing.

posted without vetting until Lee returns

(published on 01/18/2015)

Follow-Up #47: experiments on supercooling

One direct possibility is to buy some particle filters and use these to remove particles from the water. Then you can reuse the same filtered water many times. For example, 0.22 micron cellulose acetate filters should remove most dust.  I think you can buy these in small quantities and use them with a standard syringe. It would help to experiment with different types of bottles. Perhaps standard polyethylene would be good. It's a good idea to avoid glas because it can break as the water freezes.

You could also buy a few brands of bottled water and see which supercool best. Some already seem to be filtered and in suitable bottles, because our readers report accidental supercooling.

(published on 12/11/2016)

Follow-Up #48: minerals and supercooling

The minerals will slightly lower the freezing point but have very little effect on supercooling. The reverse osmosis process has the incidental effect of filtering out not just ions and molecules but also lager particles. That's what allows the supercooling to work so well, since it's those larger particles that trigger freezing.

(published on 02/05/2017)

Follow-Up #49: acetate hot ice

I can't think of any ordinary liquids that can't be supercooled at least a little bit. The liquid-solid transition in 3-D is first-order, which means that even looking at a small region the structure of the liquid and solid look different. That makes it hard for the liquid to find its way over to the different solid state, since in between it has to go through some unlikely state that's not either one. (I guess you could say the liquid 3 He can't be supercooled, however. That's because at atmospheric pressure the lowest-temperature phase is actually liquid, not solid, due to some quantum spin effects.)

From what I've just read about "hot ice" on Wikipedia, it sounds like the hot ice heaters use supersaturated solutions. The physics behind supersaturation and supercooling are quite similar, however. Getting that first little acetate crystal started is hard, just like getting that first little ice crystal started is hard. What's different is that when the phase transition is done, water forms a crystal. The supersaturated solution forms crystals together with leftover liquid.

I think there's a general correlation between the temperature of a phase transition and the latent heat. High temperature transitions tend to involve larger latent heat. It's not a rigid relation by any means, however. In general, phase transitions in any temperature range don't even have to be first-order (although liquid-solid is), which means that they don't even have to have any latent heat.

(published on 02/23/2017)

Follow-Up #50: water didn't freeze

The most likely reason is that each of the other bottles had a fleck of something in it that triggered the freezing. The last bottle maybe didn't have that, so it stayed supercooled longer.

(published on 08/03/2018)

Follow-up on this answer

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• Melting & Freezing

How to flash freeze water in a bottle

Science in sweatpants: supercool flash freeze.

Today’s experiment may be something you’ve seen on social media, we’ll be instantly freezing a bottle of water and growing ice crystals.

AUSTIN, Texas - Today’s experiment may be something you’ve seen on social media, we’ll be freezing a bottle of water and growing ice crystals instantly.

We’ll be working with supercooled water, which is water that’s cooled below the freezing point but remains in liquid form. The process we’re demonstrating today is known as nucleation. Nucleation describes the process where ice crystals begin to form around a point known as a nucleus. Once the ice crystals form around the nucleus, they’ll continue to grow throughout the liquid.

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Here’s what you’ll need:

• Room temperature, purified water (I’m using Fiji 500 mL bottled water)
• Ice cubes for the second demonstration

We’ll need purified water because it’s free from sediments and impurities. If we were using tap water or mineral water, the minerals/sediments in the water would act as nuclei and freezing would begin too soon.

To get started, place two bottles of water in the freezer and set a timer for 2 hours and 15 minutes. It’s important that these bottles are undisturbed throughout the entire 2 hours and 15 minutes. If the bottles are jostled or the freezer temperature changes too rapidly, the nucleation process could begin before we want it to.

After the timer goes off…gently remove one of the bottles from the freezer. If everything has gone according to plan, the water should still be in its liquid form.

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Experiment 1:

Now slam the bottle on the counter. The entire bottle of supercooled water should freeze within seconds. This happens because the jarring motion forces a few of the water molecules to line up into a crystal lattice structure that acts as a nucleus for the rest of the crystals to grow off of. Super cool!

Experiment 2:

Now what happens when we provide a nucleus for the ice crystals to grow on?

Take your second bottle of supercooled water out of the freezer. Pour the water over your ice cubes and watch as the water instantly freezes and creates an icy stalagmite.

That’s because the ice cubes are made up of ice crystals so when the supercooled water touches them, it instantly freezes.

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• Classical Physics

Pure water doesn't freeze at 32F ?

• Thread starter pallidin
• Start date Jun 8, 2009
• Tags Pure Water
• Jun 8, 2009

A PF Mountain

• Fluctuating hydrodynamics theory could describe chaotic many-body systems, study suggests
• X-rays from atomic systems could reveal new clues about rival quantum theories
• ATLAS probes Higgs interaction with the heaviest quarks

A PF Molecule

pallidin said: and there is nothing for that water to freeze on

A PF Planet

You may also want to check for superheated water - same problem, although on the other end of the scale. Note, that fact that supercooled and superheated water exists is not in conflict with the statements "water freezes at 0 deg C" and "water boils at 100 deg C". Both superhated and supercooled water are metastable.  

On a related note, pedants will eschew the term "freezing temperature" because of this issue: freezing involves an energy barrier, so this temperature can vary with cooling rate, impurities, container internal smoothness, etc. "Melting temperature" is better defined; there's little or no activation energy associated with melting.  

Thanks. This is making more sense now. It's funny, for all the years I've involved myself with physics(as a layman) every once-in-a-while something pops up that surprises me and I need clarification. And Mapes, I like your "related note" Thanks.  

There's an interesting experiment you can do with this - if you take high end pure bottled water and put it in a freezer for awhile, you can take it out and it won't be frozen. But, if you tap the bottle all of the water will freeze at once. It's very cool to watch.  

• Jun 9, 2009

Monocles said: There's an interesting experiment you can do with this - if you take high end pure bottled water and put it in a freezer for awhile, you can take it out and it won't be frozen. But, if you tap the bottle all of the water will freeze at once. It's very cool to watch.

pallidin said: Indeed, those are great demonstrations. However, the "freezing" does not occur all-at-once. In fact, there is a substantial lag.

• Jun 10, 2009

DaveC426913 said: BTW, I have some heat pads that do this.

Borek said: Sodium thiosulfate pentahydrate.

mgb_phys said: Do we allow that sort of language on pf? You can easily do the opposite experiment (superheating) with a cup of coffee, a microwave and a clean white shirt.

FAQ: Pure water doesn't freeze at 32F ?

1. why doesn't pure water freeze at 32f.

Pure water doesn't freeze at 32F because the freezing point of water is affected by other factors such as impurities, pressure, and surface tension. When water is pure, it lacks impurities that can act as nucleation sites for ice crystals to form, causing the freezing point to decrease.

2. What other factors can affect the freezing point of water?

Aside from impurities, pressure can also affect the freezing point of water. As pressure increases, the molecules in water are packed closer together, making it harder for them to move and form ice crystals. This results in a decrease in the freezing point of water.

3. Is the freezing point of water always 32F?

No, the freezing point of water can vary depending on the purity and other factors. For example, seawater has a lower freezing point due to the presence of salt and other minerals.

4. Does the temperature need to be exactly 32F for pure water to freeze?

No, the temperature does not need to be exactly 32F for pure water to freeze. The freezing point of water can vary depending on the purity and other factors, so it is possible for pure water to freeze at slightly different temperatures.

5. Can pure water freeze at temperatures higher than 32F?

Yes, pure water can freeze at temperatures higher than 32F. This can happen under certain conditions, such as when the water is under high pressure or when it is supercooled below its freezing point without any impurities present to act as nucleation sites.

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• Open access
• Published: 04 September 2024

Small LEA proteins mitigate air-water interface damage to fragile cryo-EM samples during plunge freezing

• Kaitlyn M. Abe   ORCID: orcid.org/0000-0001-6055-5604 1 ,
• Gan Li 1 , 2 ,
• Qixiang He   ORCID: orcid.org/0000-0002-0738-679X 1 ,
• Timothy Grant   ORCID: orcid.org/0000-0002-4855-8703 1 , 2 &
• Ci Ji Lim   ORCID: orcid.org/0000-0003-3327-1926 1  

Nature Communications volume  15 , Article number:  7705 ( 2024 ) Cite this article

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• Cryoelectron microscopy
• Structural biology
• Structure determination

Air-water interface (AWI) interactions during cryo-electron microscopy (cryo-EM) sample preparation cause significant sample loss, hindering structural biology research. Organisms like nematodes and tardigrades produce Late Embryogenesis Abundant (LEA) proteins to withstand desiccation stress. Here we show that these LEA proteins, when used as additives during plunge freezing, effectively mitigate AWI damage to fragile multi-subunit molecular samples. The resulting high-resolution cryo-EM maps are comparable to or better than those obtained using existing AWI damage mitigation methods. Cryogenic electron tomography reveals that particles are localized at specific interfaces, suggesting LEA proteins form a barrier at the AWI. This interaction may explain the observed sample-dependent preferred orientation of particles. LEA proteins offer a simple, cost-effective, and adaptable approach for cryo-EM structural biologists to overcome AWI-related sample damage, potentially revitalizing challenging projects and advancing the field of structural biology.

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Introduction.

The “resolution revolution” of cryogenic-electron microscopy (cryo-EM) marked a significant shift in the field of structural biology 1 . However, the continued growth of cryo-EM single-particle analysis faces a critical problem, namely sample damage that occurs during grid preparation 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . The standard method for cryo-EM single-particle analysis sample preparation is plunge freezing, where small volumes of sample are applied onto a pretreated cryo-EM grid, blotted, and rapidly plunged into a cryogen such as liquid ethane (Fig.  1a ). This process forms a thin layer (~10–100 nm) 5 , 10 of sample-embedded vitreous ice for transmission electron microscopy imaging 11 , 12 (Fig.  1b ). Between the blotting and rapid freezing steps, the sample exists as a thin aqueous film with a high surface-to-volume ratio. During this transitional phase (typically lasting for seconds), the proteins in the aqueous solution collide with the air–water interface (AWI) thousands of times before finally being vitrified 5 , 9 .

Cartoon illustration of the process and outcomes of preparing cryo-EM SPA grids and the hypothesized effects of LEA proteins on sample structural integrity. a Shows a Cryo-EM SPA grid preparation robot alongside a detailed view of a typical holey grid with vitrified ice, which is used for embedding protein samples. b – d Depict different states of protein sample distribution within the vitrified ice. In panel ( b ), an ideal sample distribution is shown where proteins are evenly dispersed without any structural damage. c Illustrates the typical impact of air–water interface (AWI) damage on protein samples, leading to preferred orientation, complex dissociation, and protein denaturation. d Demonstrates how LEA proteins can mitigate sample damage by forming a barrier at the AWI, which significantly mitigates these damages and preserves protein integrity in vitrified ice.

Adsorption to the AWI can destroy the protein’s structural integrity, causing it to denature, or disintegrate 2 , 5 , 7 , 13 (Fig.  1c ). In a scenario where the protein maintains its structure after AWI adsorption, the sample interaction with the AWI may be biased towards certain regions, leading to preferred sample orientations and an anisotropic 3D reconstruction of the sample 2 , 3 , 14 , 15 . To mitigate the AWI problem, researchers have developed a variety of solutions. These include the addition of mild detergents to the sample 16 , 17 , using chemical crosslinking 18 , 19 , 20 , 21 , and adsorbing or tethering samples to surfaces to prevent sample contact with the AWI 4 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 . More sophisticated techniques involve rapidly freezing the sample before the proteins can diffuse to the AWIs 30 , 31 , 32 , 33 , 34 , 35 . However, these methods are often technically challenging, cost-prohibitive and demonstrate sample-specific results.

Here we show an AWI mitigation strategy that is accessible, cost-effective, and readily deployable in all cryo-EM facilities and research laboratories.

AavLEA1 mitigates AWI damage to fragile cryo-EM samples during grid plunge freezing

In search of a new solution to mitigate AWI damage, we explored the biological world, hypothesizing that cellular response mechanisms to desiccation stress could offer an evolved solution for mitigating protein damage caused by AWI interaction. We identified late embryogenesis abundant (LEA) proteins 36 , 37 , 38 , specifically a group 3 LEA protein, AavLEA1, from the roundworm (nematode) Aphelenchus avenae 39 as a potential candidate. Studies have indicated that AavLEA1, which has a molecular weight of ~16 kDa, prevents protein aggregation due to desiccation 39 . This is likely due to the formation of an AavLEA1 barrier which mitigates damage caused by direct interaction of the sample with the AWI 38 . We thus propose that AavLEA1 can be used as a sample additive to mitigate damage to protein samples caused by AWI interaction during cryo-EM grid plunge freezing (Fig.  1d ). The relatively small molecular mass of AavLEA1 (approximately 16 kDa) compared to a typical sample of interest (several hundred kDa) should allow AavLEA1 to reach the AWI faster than the larger samples.

To evaluate the AWI damage mitigation effects of LEA proteins, we used two protein complexes that are AWI-sensitive as model systems: human DNA polymerase-α-primase (PP) 40 , 41 , 42 and Polycomb repressive complex 2 (PRC2) 20 , 43 , 44 . It is important to note that in this study, both complexes were tested in their apo-state, and all data collection was performed using a 200 keV electron microscope with a direct electron detector (for details, please see the Methods section). Reported cryo-EM structures of apo-state PP complexes were solved using chemically crosslinked samples 42 , suggesting that the PP particles fell apart due to AWI damage during plunge freezing. Another arguably more challenging structure to solve for cryo-EM single-particle analysis is that of PRC2. Reported PRC2 cryo-EM structures were solved by highly specialized strategies using either chemically crosslinked samples on thin carbon-support grids 20 , 45 or biotinylated samples tethered onto Streptavidin-crystal grids 43 , 44 .

We found that the addition of AavLEA1 to PP and PRC2 before freezing eliminates the need for the abovementioned methods, allowing us to solve their structures at comparable or higher resolutions (Fig.  2 ). The optimal addition amount was found to be between 1:8–1:40 molar ratio of the sample to the LEA proteins (Supplementary Figs.  1 – 3 ). The addition of AavLEA1 at a 1:40 PP:AavLEA1 molar ratio before plunge freezing led to considerably better grids than freezing the PP sample alone; with clear and homogeneously sized particles (Fig.  2a ). Multiple high-resolution two-dimensional (2D) class averages of PP were obtained from these particles (Fig.  2b ), and subsequent image processing led to a 3.0 Å global resolution map of the apo-state PP (Fig.  2c and Supplementary Fig.  3 ). This is the highest resolution cryo-EM map reported for the human apo-state PP; the previous X-ray structure was solved at 3.6 Å 46 and a chemically crosslinked cryo-EM structure determined at a 3.8 Å global resolution 42 . Most importantly, we demonstrated that AavLEA1 can be used to mitigate sample damage from AWI interactions during plunge freezing, resulting in high-resolution cryo-EM maps.

Cryo-EM single-particle analysis of human polymerase α-primase complex (PP), ( a – c ), and polycomb repressive complex 2 (PRC2), panels ( d – f ), both with the addition of Nematode AavLEA1. a , d Display representative micrographs and CTFs-cropped up to the ice ring for the complexes both alone and with AavLEA1 added at a 1:40 ratio, highlighting improved sample preservation due to LEA protein addition. Representative micrographs were chosen out of 42 ( a ) top, 4403 ( a ) bottom, 5 ( d ) top, and 2843 ( d ) bottom. b , e Depict 2D class averages, illustrating defined and consistent particle shapes with visible protein features when AavLEA1 was used. Finally, c , f show reconstructed cryo-EM maps of the complexes from the AavLEA1 datasets, presented in two orientations.

Initially, we had some potential concerns with the AavLEA1 approach. Firstly, the addition of AavLEA1 could have led to an elevated background signal in the micrograph. Secondly, interaction with AavLEA1 could have distorted the sample conformation. Given that we obtained a 3.0 Å resolution cryo-EM map for human PP apo-state complex in the presence of excess AavLEA1 (sample:AavLEA1 of 1:40), we deduce that background AavLEA1 did not significantly affect image alignment or hinder high-resolution reconstruction. Additionally, we did not find any extra map density that would indicate AavLEA1 binding to PP in a consistent manner, nor a significant change in the PP conformation; we calculated a RMSD of 1.2 Å when a refined model derived from the 3.0 Å cryo-EM map was aligned to a published crystal structure of an apo-state human PP (PDB: 5EXR) 46 (Supplementary Figs.  4 , 5 and Table  1 ). In short, AavLEA1 addition did not alter the PP apo-state conformation.

We saw similar results for PRC2 with the addition of AavLEA1 (1:40 PRC2:AavLEA1 molar ratio)—Adding AavLEA1 resulted in clear and homogeneously sized particles (Fig.  2d ). Without additives, we saw smaller sized particles in the micrographs, indicating broken PRC2 complexes, consistent with previous studies 47 . Subsequent cryo-EM image processing led to a 3.8 Å global resolution map of PRC2 (Fig.  2e , f and Supplementary Fig.  6 ). This result further exemplifies the efficacy of AavLEA1 in facilitating the determination of high-resolution cryo-EM structures of protein samples sensitive to the air–water interface.

A truncated form of LEA protein from tardigrade also mitigates AWI sample damage

Encouraged by the success of using nematode AavLEA1 to mitigate AWI sample damage, we pondered whether LEA proteins from other organisms could provide comparable results. Tardigrades, also known as water bears, are tolerant of desiccation stress 48 , and the species Ramazzottius varieornatus has a group 3 LEA protein, RvLEAM 49 . Because RvLEAM has nine LEA-like motifs and is about 30 kDa in mass, we truncated it to ~15 kDa to increase its diffusion coefficient and match the size of AavLEA1 for ease of comparison (refer to the Methods section for details). The truncated RvLEAM is hereafter termed RvLEAM short .

Similar to AavLEA1, adding RvLEAM short to PP or PRC2 sample at a molar ratio of 1:6 sample:RvLEAM short before plunge freezing led to the appearance of monodisperse and discernable particles (Fig.  3 ). From datasets collected over a single night, we successfully reconstructed cryo-EM maps of human apo-state PP and PRC2 at a reported global resolution of 4.5 and 3.7 Å, respectively (Fig.  3c, f and Supplementary Figs.  7 , 8). The final resolution of PP and RvLEAM short in this dataset is lower than our other PP dataset because significantly fewer movies were collected (see methods). Given that this is the second LEA protein we have tested that successfully rescued the cryo-EM structure determination of two challenging AWI-sensitive protein complexes, we believe group 3 LEA proteins, as a cryo-EM single-particle analysis sample additive, offers a promising and powerful method for structural biologists to mitigate sample damage caused by AWI during plunge freezing.

Cryo-EM single-particle analysis of protein complexes with RvLEAM short . a , d Display representative micrographs and CTFs-cropped up to the ice ring-out of the 1308 and 3896 collected respectively of the polymerase-primase complex (PP) and Polycomb repressive complex 2 (PRC2), each treated with RvLEAM short at a molar ratio of 1:6. b , e Show 2D class averages, which demonstrate the structural homogeneity and quality of the sample with RvLEAM short added. c , f Present the reconstructed cryo-EM maps of PP and PRC2, depicted in two orientations, achieving resolutions of 4.5 and 3.7 Å, respectively.

Samples with LEA protein addition distribute at vitrified ice surfaces

While our prediction that LEA proteins mitigate AWI damage was correct, our initial hypothesis about the mechanism of LEA-mediated AWI damage mitigation proved inaccurate. We speculated that once the LEA proteins formed a barrier at the AWI, which we termed the LEA-water interface (LWI), the sample particles would remain in the aqueous layer. When imaged in vitreous ice after plunge freezing, we expected the projected views of the frozen particles to be randomly distributed (Fig.  1 ). However, a major underlying assumption was that there was no interaction between the sample and the LWI. If such interactions exist, they would manifest as a bias in the particle orientation (view projection) 6 , 17 . Our particle orientation distribution analysis for the cryo-EM maps derived from the LEA protein datasets suggests this was the case (Fig.  4a and Supplementary Table  2 ). Calculations from the 3DFSC server 14 indicated that the PP maps show a good isotropic distribution, approaching perfect isotropy with a sphericity of 0.98 for those with AavLEA1 and 0.83 with RvLEAM short . Conversely, the PRC2 maps exhibited poorer particle orientation distribution, regardless of the LEA protein used; sphericity values were 0.77 and 0.79 for maps with AavLEA1 and RvLEAM short , respectively. Overall, these results suggest that the samples interact with the LWI to varying degrees, dependent on the specific sample.

Particle orientation distribution and cryo-electron tomography (cryo-ET) analysis for samples with LEA proteins addition. a Displays Mollweide projections that compare the particle distribution for the polymerase α-primase complex (PP) and Polycomb repressive complex 2 (PRC2) with AavLEA1 (1:40) and RvLEAM short (1:6) added respectively. Corresponding sphericity values demonstrate the degree of isotropy achieved in the cryo-EM map under each condition. b , c Show cryo-ET cross-sectional analysis of the spatial distribution of PP and PRC2 particles within the grid holes, respectively. These plots highlight the location of particles relative to the edge of the holes and identify regions affected by ice contamination. The axes are expressed in pixels, with a scale of 4.4 Å per pixel.

If the samples are indeed interacting with the interface, the particles should be distributed as plane(s). To test this, we used fiducial-less cryo-electron tomography (cryo-ET) analysis to visualize the particle distribution of PP with AavLEA1 (1:40 molar ratio) and PRC2 with RvLEAM short (1:6 molar ratio) in our grids (Fig.  4b , c and Supplementary Movies  1 , 2 ). In both cases, we saw most of the particles were distributed within one or two planes in the vitrified ice, confirming that the samples were adsorbed to a surface in the presence of LEA proteins.

Due to the small sizes of LEA proteins, we were unable to visualize them in our cryo-EM images directly, preventing us from determining their empirical coverage at the AWI. Consequently, we cannot rule out the possibility of incomplete LEA protein coverage, and that the particle orientation bias could have resulted, in part or entirely, from interactions with exposed pockets of the AWI. If this is indeed the case, it is reassuring to note that these interactions with the AWI, in the presence of LEA proteins, did not significantly compromise the samples’ structural integrity, as demonstrated by the reconstructed high-resolution cryo-EM maps of the tested samples (Figs.  2 , 3 ).

Biochemical strategies to improve particle orientation distribution of LEA datasets

Given that interactions between the sample and LEA proteins at the LWI can contribute to the observed particle orientation bias, we aimed to explore strategies that influenced these interactions to achieve a desirable particle orientation outcome. Both AavLEA1 and RvLEAM short are predicted to form amphiphilic alpha-helical structures, with the hydrophobic face oriented towards the air at the AWI and the hydrophilic side (negatively charged) facing the aqueous solution 49 , 50 . Thus, the negatively charged, solution-facing side of the LWI could create a “sticky” surface that leads to the particle orientation bias observed in the LEA datasets.

Past studies have demonstrated that electrostatic charge on a surface, whether AWI or carbon film, can influence sample particle orientation 51 , 52 . Using PRC2 and RvLEAM short as the model condition, we added a divalent cation, MgCl 2 , to the sample buffer to neutralize the negatively charged LWI surface, with the hope that this strategy would change the PRC2 particle orientation distribution. However, this approach did not improve the particle orientation distribution and instead made it worse with more limited views (Supplementary Fig.  9 ).

Next, we investigated the effect of mild glutaraldehyde crosslinking on PRC2 prior to freezing with RvLEAM short to determine if this could improve particle orientation distribution. Our rationale was that the glutaraldehyde reaction would neutralize the positively charged groups on the surface of PRC2 and lead to particle orientation distribution changes, as has been demonstrated by past studies 18 , 19 , 52 . Chemical crosslinking also has the benefit of stabilizing the complex for improved structural homogeneity 18 , 19 . We collected cryo-EM datasets of crosslinked PRC2 with RvLEAM short at a 1:6 sample:LEA molar ratio; the PRC2 samples were crosslinked with glutaraldehyde for 2 and 10 min (Supplementary Fig.  10 ).

Our analysis indicated that chemical crosslinking improved the orientation distribution of PRC2 particles (Fig.  5a and Supplementary Figs.  11 , 12 ). The map sphericity increased from 0.79 in non-crosslinked samples to 0.83 after 2 min of crosslinking. Extending the crosslinking to 10 min further enhanced this value to 0.97 (Supplementary Table  2 ). This incremental improvement suggests that longer crosslinking durations may be slightly more effective. The 10 min crosslinked sample dataset enabled us to achieve a 3.1 Å global resolution cryo-EM map of the PRC2 complex (Fig.  5b ). This map represents the highest resolution yet reported for the human PRC2 apo-state complex. Given that the map was reconstructed from a dataset collected on a 200 kV microscope, using a 300 kV microscope could potentially yield an even higher resolution. Our PRC2 model, built using this cryo-EM map, has a structure similar to that obtained through the streptavidin-biotin affinity EM grid approach 25 , 44 , with the RMSD between the two aligned structures being 0.89 Å (Supplementary Figs.  13 , 14 and Supplementary Table  3 ).

a Displays Mollweide projections of the particle orientation distribution for the Polycomb repressive complex 2 (PRC2) treated with chemical crosslinking for 2 and 10 min, demonstrating improved isotropy as evidenced by the increased sphericity values of 0.83 and 0.97, respectively. b Shows the high-resolution cryo-EM maps of PRC2 crosslinked for 10 min, presented in two orientations to highlight the detailed structural features achieved, with a global resolution of 3.1 Å.

A control 10 min crosslinked PRC2 cryo-EM dataset without RvLEAM short was also collected to determine the effect of adding LEA proteins crosslinked samples. While discernable and somewhat homogeneous particles were observed, the subsequent single-particle analysis revealed that most particles were broken subcomplexes of PRC2 (Supplementary Fig.  15 ), a result consistent with a previous study 47 . Nonetheless, we obtained a 4.3 Å cryo-EM map of an intact PRC2 complex (Supplementary Fig.  15 ), although the efficiency was modest; from 3274 movies, ~1.7 million particles were picked, but only about ~3% contributed to the final cryo-EM map reconstruction (Supplementary Fig.  15 ). In contrast, with the addition of RvLEAM short , ~14% of initially picked particles (~2.5 million particles) were utilized in the final reconstruction (Supplementary Fig.  12 ). Furthermore, particles from the crosslinked control dataset suffered from biased particle orientation, with a 3DFSC calculated sphericity value of 0.66. The inclusion of RvLEAM short improved the sphericity value to 0.97 (Supplementary Table  2 ). Overall, the above comparison suggests the addition of RvLEAM short enhanced the stability and particle orientation distribution of crosslinked PRC2 complexes.

Comparative analysis of LEA proteins and CHAPSO as AWI damage mitigation strategies

CHAPSO, a zwitterionic detergent, can mitigate AWI damage to protein samples during plunge freezing 17 , 53 . In our hands, it is an effective strategy for determining the high-resolution cryo-EM structures of PP-related complexes 40 , 54 . Hence, we wanted to compare LEA protein’s efficiency in obtaining a high-resolution cryo-EM structure against that of CHAPSO. To this end, we determined a 3.4 Å cryo-EM map of apo-state PP using CHAPSO (Fig.  6a and Supplementary Fig.  16 ). By visual inspection, we found no discernable differences between the two cryo-EM maps or modeled structures (compared to the 40:1 AavLEA1:PP cryo-EM map, Supplementary Figs.  17 , 18 and Table  1 ). However, three separate empirical analyses, Reslog 55 (Fig.  6b ), per-particle spectral signal-to-noise (ppSSNR) 56 (Fig.  6c ), and Rosenthal–Henderson 57 (Supplementary Fig.  19 ) plots, all pointed to the AavLEA1 dataset having the better particle image quality. It is possible that thicker ice in the CHAPSO dataset impacted particle image quality, explaining the observed differences. Nonetheless, our results demonstrate that AavLEA1, when used as a sample additive, can achieve cryo-EM map quality comparable to, or even better than, that obtained with detergents.

a Showcases the reconstructed cryo-EM map of the polymerase α-primase complex, visualized in two orientations, achieving a global resolution of 3.4 Å. b , c Detail the ResLog and per-particle spectra SNR (ppSSNR) analyses respectively, comparing the effects of AavLEA1 and CHAPSO addition on particle image data and map reconstruction quality. The ResLog analysis in panel ( b ) illustrates the spatial frequency improvements associated with each additive, plotted against batch size on a logarithmic scale, indicating that AavLEA1 outperforms CHAPSO at higher spatial frequencies. c Displays the logarithm of ppSSNR, demonstrating that AavLEA1 maintains higher SNR values across the majority of the spatial frequencies tested. Source data are provided as a Source Data file.

A major advantage of using AavLEA1 over CHAPSO, or other detergents, is its effectiveness when cryo-EM samples are limited or unstable at high concentrations. CHAPSO and similar detergents typically require high sample concentrations 51 , 58 —over 4 mg/mL or ~13 μM for a 300 kDa protein sample—which can be challenging for precious samples. In contrast, with AavLEA1, we used only 1–1.5 μM of sample protein, which is more than ten times less than that required for CHAPSO or detergents in general. While the application of LEA proteins can lead to an anisotropic distribution of particle orientations (see Result section: Samples with LEA proteins addition distribute at vitrified ice surfaces and Fig.  4b, c ), this issue does not arise with the use of CHAPSO. The particles in the CHAPSO dataset have an even distribution of particle orientations (Supplementary Fig.  16 and Table  2 ). Consequently, the calculated sphericity value of the PP cryo-EM map from the CHAPSO dataset is 0.99.

In summary, we have shown that two group three LEA proteins, AavLEA1 from nematodes and RvLEAM (truncated) from tardigrades, can mitigate sample damage caused by sample interaction with the AWI during cryo-EM plunge freezing. We demonstrated the effects on two model multi-subunit protein complexes, human PP and PRC2, in the apo-state, both challenging targets for cryo-EM analysis. By simply adding AavLEA1 or RvLEAM short to the samples before plunge freezing, we determined the cryo-EM structures of both PP and PRC2 at comparable, or better resolution than those previously reported using more challenging and complicated anti-AWI damage strategies 20 , 42 , 43 , 44 , 47 . It is important to note that these fragile samples did not yield any discernable monodisperse particles using standard plunge freezing methods. Therefore, our results underscore the effectiveness of LEA proteins in revitalizing cryo-EM projects that would otherwise be deemed unfeasible. While our study demonstrates the effectiveness of LEA proteins in mitigating AWI damage for PP and PRC2 complexes, further investigation is needed to determine their applicability to other macromolecular assemblies, membrane proteins, or smaller protein complexes.

The LEA proteins require lower sample concentration compared to other AWI mitigation solutions, such as Spotiton 31 , 32 , 33 or detergents 51 , 58 , 59 , offering a significant advantage when working with limited samples or those prone to aggregation at high concentrations. Another common solution for accommodating low sample concentrations is the use of cryo-EM grids with support films. However, the quality of these specialized EM grids can vary due to production batch reproducibility, and they can be technically challenging to produce in research laboratories. Unlike these methods, the application of LEA proteins does not require specialized grids and is compatible with standard cryo-EM holey grids. While the utilization of LEA proteins can lead to preferred particle orientation problems and varying degrees of anisotropy depending on the sample, this issue is not insurmountable. It can be mitigated by employing chemical crosslinking 18 or tilted-stage data collection strategies 14 . Additionally, given that the proteins adhere to LEA-water interfaces, the molar ratio between the sample and LEA proteins may not be a key optimization parameter for sample grid plunge freezing. Rather, the concentration of LEA proteins used is important. In our hands, a minimum of 6 µM LEA proteins was sufficient to mitigate AWI damage to fragile samples.

We believe that LEA proteins represent a promising avenue for structural biologists to revisit cryo-EM projects previously hindered by AWI issues, particularly those that have exhausted conventional AWI mitigation strategies. These proteins can be produced in large quantities using standard bacterial expression systems and purification schemes, providing a sustainable and cost-effective alternative to methods that rely on more expensive and perishable materials and reagents. Most importantly, the accessibility and economic benefits of LEA proteins enable any structural biology laboratory or cryo-EM facility to readily adopt this method, potentially revolutionizing their approach to cryo-EM structure determination.

Expression and purification of AavLEA1 and RvLEAM short

The expression plasmid for HIS-tagged AavLEA1 was sourced from the Addgene plasmid repository [pET15b-AavLEA1, a gift from Claude Férec (Addgene plasmid # 53093)] 60 . The expression plasmid for HIS-tagged RvLEAM short was constructed by inserting a truncated cDNA from pEThT-RvLEAM [pEThT-RvLEAM was a gift from Takekazu Kunieda (Addgene plasmid # 90033)] 49 into a pET15b vector. RvLEAM short encodes residues 58-181 of RvLEAM (A0A0E4AVP3.1). Both recombinant AavLEA1 and RvLEAM short were expressed in Escherichia coli BL21 (DE3) cells. A single bacterial colony containing the transformed plasmid was cultured overnight in 2 mL of Luria Bertani broth (LB) with 100 µg mL −1 carbenicillin at 37 °C. This starter culture was then used to inoculate 1 L of LB supplemented with the same antibiotic. At an optical density (A600) of 0.6, gene expression was induced using 0.1 mM isopropyl-β- d -thiogalactopyranoside (IPTG) for 16 h at 12 °C and shaken at 230 rpm.

Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 10 mM imidazole, 1 mM DTT or TCEP, 1 mM PMSF), and lysed via sonication. The cell debris was then removed by centrifugation. The clarified lysate was incubated with pre-equilibrated nickel-NTA resin (Qiagen, Germany) and stirred for 1 h at 4 °C. The protein-bound resin was washed three times with 50 mL of lysis buffer. Proteins were eluted with 10 mL of elution buffer (wash buffer supplemented with 250 mM imidazole) using a gravity flow column. The proteins were then concentrated to ~500 μL using a 3 kDa MWCO spin column and further purified on a Superdex 75 10/300 size-exclusion chromatography (SEC) column (Cytiva, USA) pre-equilibrated with SEC buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 1 mM TCEP, 10% glycerol). Eluted fractions were analyzed by SDS-PAGE. Chosen fractions were pooled, concentrated, snap-frozen in 5–10 µL aliquots, and stored at −80 °C until use. The protein concentration of the aliquots was determined using the Beer–Lambert equation, with absorbance measurements obtained from a NanoDrop spectrophotometer (Thermo Fisher, USA) and extinction coefficients calculated based on their protein sequences.

Production of recombinant human DNA polymerase alpha-primase

Recombinant human Polα–primase was expressed and purified as previously described 54 . Briefly, Trichoplusia ni ( T.ni ) cells (Expression System) were infected with four baculoviruses (POLA1, POLA2, PRIM1, and PRIM2) for the co-expression of human Polα–primase. The infected T.ni cells were collected for protein purification after 66–68 h post-infection. The human Polα–primase was obtained using a tandem affinity approach. First, His-tagged POLA2, PRIM1, and PRIM2 were captured using Ni-NTA agarose resin (Qiagen). The elute was then subjected to a second pull-down using Strep-Tactin XT 4Flow-resin (IBA LifeScience) for strep-tagged POLA1. The purified Polα–primase complex was verified using SDS-PAGE analysis.

Production of recombinant human polycomb repressive complex 2

Purified recombinant human polycomb repressive complex 2 (PRC2) protein complexes were generously provided by Dr. Tom Cech at the University of Colorado Boulder 44 .

PRC2 cryo-EM sample glutaraldehyde crosslinking

Approximately 2 µM of PRC2 was incubated with a 0.1% (v/v) final concentration of glutaraldehyde for 2- and 10-min intervals. After each interval, an aliquot was removed and quenched with 80 mM Tris-HCl to stop the reaction. SDS-PAGE was then used to assess the crosslinking efficiency of the PRC2 samples at each incubation time point.

All samples were thawed just prior to cryo-EM grid preparation. Holey carbon cryo-EM grids, either Quantifoil R 1.2/1.3 300 mesh Au or C-flat R 1.2/1.3 300 mesh Au, were glow discharged using a PELCO EasiGlow glow-discharge unit (15 mA for 30 s with a 10-s hold). These treated grids were used within 30 minutes. Protein samples were diluted to the working concentration immediately before application to the grid. Where indicated, the sample (~3.5 μL) was supplemented with LEA proteins, CHAPSO, or MgCl 2 just before being applied to the glow-discharged grid. Typically, the high-concentration LEA protein stock was first diluted to an intermediate working solution using the sample buffer and then mixed with the sample (e.g., 2 μL of LEA with 2 μL of the sample). The grid was then blotted for 4 to 6 s at 4 °C and 95% humidity before being plunged frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher, USA).

For all conditions except those specified, 1–1.5 μM of PP or PRC2 were used with the indicated molar ratio of AavLEA1 or RvLEAMshort. For conditions with PP and 4 mM CHAPSO, 13.8 μM of PP was utilized.

Cryo-EM data collection

All data collections and screenings were conducted on a Talos Arctica 200 kV TEM (Thermo Fisher Scientific, USA) equipped with a Gatan BioQuantum K3 direct electron detector (Gatan, USA). Data screening and acquisition were managed using either EPU (Thermo Fisher, USA) or SerialEM 61 . All cryo-EM datasets were collected at a pixel size of 1.064 Å/pixel, with a total dose of 50 e − Å −2 distributed across 40 frames. The CDS counting mode was utilized along with a 20 eV energy filter slit. The defocus range was set between −1 and −2.5 μm in 0.25-μm intervals.

Cryo-EM data processing

For all datasets, image processing was carried out using cryoSPARC 62 . In brief, movies were subjected to patch motion correction, and the aligned micrographs had their contrast transfer function (CTF) estimated. The CTF values were utilized to select a subset of micrographs deemed suitable for high-resolution single-particle analysis. Detailed procedures for subsequent image processing steps specific to each dataset are outlined below:

1.5 μM PP, 12 μM AavLEA1 (1:8) dataset

A total of 2764 movies were collected. After micrograph curation 2112 movies remained and 1,724,984 particles were extracted and binned 4x binning (4.3 Å/pixel). After 2D classification, 785,653 particles proceeded to ab initio reconstruction and were sorted into four separated reference-free 3D classes. Particles underwent another round of ab initio modeling and separated into two classes. The intact complex was re-extracted at 1.1 Å/pixel and was sorted into one of the two classes (239,029 particles, 75%). Non-uniform refinement of this class with per-particle CTF refinement resulted in a global resolution (reported at Fourier shell correlation of 0.143) of 3.6 Å.

1.5 μM PP, 60 μM AavLEA1 (1:40) dataset

A total of 4403 movies were collected. After micrograph curation, 500 movies were initially used, with a total of 340,430 particles extracted at 4x binning. From 2D classification, 201,337 particles were selected and re-extracted at the original pixel size. Particles then proceeded to ab initio reconstruction and were sorted into four separated reference-free 3D classes. Two of the four classes resulted in intact particles, which were verified through non-uniform refinement of the combined classes using 165,472 particles, resulting in a 3.8 Å structure. Particles were extracted from the remaining 3771 movies and binned 4x, resulting in 2,668,602 particles. These particles were then sorted into 2D classes, and the selected 1,438,074 particles underwent ab initio modeling. Selected particles underwent another round of ab initio modeling with two classes. One of the two classes showed intact particles, and those 1,009,026 particles were sent to non-uniform refinement yielding a 3.4 Å global resolution. CryoSparc global and local CTF refinement jobs were run, followed by further filtering, resulting in 988,417 particles. These particles were then extracted at the original pixel size, 1.1 Å/pixel. All resulting particles underwent non-uniform refinement, reference motion correction, and heterogeneous refinement. 856,205 particles were used for a final non-uniform refinement, with a final global resolution of 3.0 Å.

1 µM PP, 6 µM RvLEAM short (1:6) dataset

A total of 1308 movies were collected. Following micrograph curation, 335,907 particles were extracted from 1076 micrographs and binned 4x. About 125,231 particles were extracted with 2D classification. These particles then proceeded to ab initio reconstruction and split into 3D classes. The selected 74,198 particles were re-extracted at the original pixel size (1.1 Å/pixel) from 1071 micrographs. Particles underwent another round of 2D classification and ab initio 3D reconstruction. Selected particles underwent non-uniform refinement and had a final global resolution of 4.5 Å. The final resolution of this dataset is lower than our other PP datasets which is likely because this data collection contains only 1308 movies while other PP datasets have 2700 or more movies.

13.8 μM PP, 4 mM CHAPSO dataset

A total of 4567 movies were collected. Following micrograph curation, 1,832,455 particles were extracted from 4510 micrographs and binned 4x. Particles underwent 2D classification where and ab initio reconstruction. Selected particles were re-extracted at the original pixel size of 1.1 Å/pixel (744,824 particles). Extracted particles were then sorted into 2D classes and underwent non-uniform refinement with per-particle CTF refinement and reference motion correction, with a final global resolution of 3.4 Å with 674,793 particles.

1.5 μM PRC2, 60 μM AavLEA1 (1:40) dataset

A total of 2843 movies were collected. Following micrograph curation, 687,121 particles were extracted from 1642 micrographs and binned 4x. Particles underwent 2D classification and ab initio reconstruction. Selected classes were re-extracted at the original pixel size, 1.1 Å/pixel, yielding 150,359 particles. The re-extracted particles underwent non-uniform refinement with per-particle CTF refinement and resulted in a global resolution of 3.8 Å.

1 μM PRC2, 6.7 μM RvLEAM short (~1:6) dataset

A total of 3896 movies were collected. Following micrograph curation, 1,766,944 particles were extracted from 3637 micrographs and binned 4x. Particles were sorted into 2D classes followed by 3D classes via ab initio reconstruction. The selected 547,610 particles were re-extracted at 1.1 Å/pixel and subjected to ab initio 3D reconstruction. The final 206,807 particles underwent non-uniform refinement with per-particle CTF refinement, resulting in a global resolution of 3.7 Å.

1 μM PRC2, 6.7 μM RvLEAM short (~1:6) 10 mM MgCl 2 dataset

A total of 4333 movies were collected. Following micrograph curation, 1,755,138 particles were extracted from 3759 micrographs and binned 4x. Particles underwent two rounds of 2D classification. From here, 408,373 particles were selected and proceeded to ab initio 3D reconstruction. The resulting 406,148 particles were re-extracted from 3746 movies at the original pixel size (1.1 Å/pixel) and underwent another round of ab initio reconstruction. The final 102,181 particles proceeded to non-uniform refinement with a final global resolution was 4.2 Å.

1 μM PRC2 crosslinked 2 min, 6.7 μM RvLEAM short (~1:6) dataset

A total of 2981 movies were collected. Following micrograph curation, 982,264 particles were extracted from 2189 movies and binned 4x. Particles were sorted into 2D classes followed by 3D classes via ab initio reconstruction. Selected particles were re-extracted at the original pixel size (1.1 Å/pixel) and underwent ab initio reconstruction again. The resulting 534,068 particles were subjected to non-uniform refinement with per-particle CTF refinement, resulting in a global resolution of 3.5 Å.

1 μM PRC2 crosslinked 10 min, 6.7 μM RvLEAM short (~1:6) dataset

A total of 3432 movies were collected. Following micrograph curation, 2,084,816 particles were extracted from 3295 movies and binned 4x. Particles underwent 2D classification followed by ab initio 3D reconstruction. Selected particles were re-extracted at the original pixel size (1.1 Å/pixel). Particles underwent non-uniform refinement, resulting in a global resolution of 3.3 Å. Global CTF refinement, reference-based motion correction, and heterogeneous refinement jobs were run. 366,459 particles were used in this final round of non-uniform refinement, resulting in a global resolution of 3.1 Å.

1 μM PRC2 crosslinked 10 min dataset

A total of 3274 movies were collected. Following micrograph curation, 1,231,903 particles were extracted from 3274 movies and binned 4x. Particles underwent 2D classification followed by ab initio 3D reconstruction. Selected particles were re-extracted at the original pixel size (1.1 Å/pixel) and underwent a second batch of ab initio 3D reconstruction, reference-based motion correction non-uniform refinement. The final 51,494 particles resulted in a global resolution of 4.3 Å.

Cryo-EM structure modeling and refinement

The published apo-state models of Polymerase alpha-primase (PP, PDB: 5EXR) 46 and Polycomb Repressive Complex 2 (PRC2, PDB: 8FYH) 44 were used as initial models for real-space refinement against their respective cryo-EM maps using Phenix 63 . Structural alignments between the published models and the refined models were performed using the MatchMaker module in ChimeraX 64 . Refinement statistics and validation reports are provided in Supplementary Tables  1 , 3 for PP and PRC2, respectively. Q-score 65 analysis was conducted for each refined model and reported in the abovementioned tables.

Particle image quality and orientation distribution analysis

ResLog plots were generated using the ResLog job in cryoSPARC 62 . Rosenthal–Henderson plots were derived from the data utilized in the ResLog plot, following the method described by Rosenthal and Henderson 57 . The per-particle spectral signal-to-noise ratio (ppSSNR) plots were produced using the FSC_noisesub data from the ResLog Analysis job in cryoSPARC. Particles were segmented into stacks of 30,000, 60,000, 90,000, 120,000, and 150,000 for analysis. The ppSSNR calculations are performed as described by ref. 66 . Cryo-EM map sphericity values are calculated using the 3DFSC server 14 . The conical FSC area ratio (cFAR) and sampling compensation factor (SCF) 56 , 67 were computed using CryoSPARC 62 .

Tilt-series collection

Tilt series were collected on a Titan Krios (Thermo Fisher Scientific, USA) operating at 300 kV, equipped with a K3 summit direct electron detector and a Quantum energy filter (Gatan, USA), controlled by SerialEM 61 . Images were collected with an exposure of 8 e-/pixel/s on the detector, with the camera operating in CDS mode, with a calibrated pixel size of 1.1 Å per pixel. Tilt series were collected using a dose-symmetric tilt scheme from −45° to 45° with a tilt increment of 3° and nominal defocus between 2 and 4 μm 68 . Each tilt angle was collected as a five-frame movie, with an exposure of 5 e – /Å 2 per tilt, and, therefore, a total exposure of 150 e – /Å 2 per tilt series.

Tilt-series data processing

Each movie was whole frame aligned using the Unblur package in cisTEM 69 . Tilt series were aligned and reconstructed at a binning factor 4 using AreTomo 70 . To further enhance the contrast of protein particles for better localization, the reconstructed tomograms were denoised using IsoNet 71 . The reconstructions are shown in Supplementary Movies  1 , 2 . These movies were created using 3dmod from the IMOD package 72 and ImageJ 73 .

Particle localization and ice surface estimation

Particles in the tomograms were picked manually with Dynamo 74 . The surfaces of vitrified ice were determined via three markers: crystalline ice contaminations above the ice, carbon edges, and the protein layers, which are assumed to be close to the surface.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The described cryo-EM maps and coordinate files have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank (PDB) under the following accession codes: Polymerase alpha-primase (PP)–AavLEA1, 1:8 molar ratio under code EMD-43619 , PP–AavLEA1, 1:40 molar ratio under codes PDB-ID 8VY3 and EMD-43628 , PP–RvLEAM short , 1:6 molar ratio under code EMD-43626 , PP–CHAPSO 4 mM under codes PDB-ID 9C8V and EMD-43627 , Polycomb repressive complex 2 (PRC2)–AavLEA1, 1:40 molar ratio under code EMD-43620 , PRC2–RvLEAM short , 1:6 molar ratio under code EMD-43621 , PRC2–RvLEAM short , 1:6 molar ratio, 10 mM MgCl 2 under code EMD-43622 , PRC2–RvLEAM short , 1:6 molar ratio, 2 min crosslink under code EMD-43623 , PRC2–RvLEAM short , 1:6 molar ratio, 10 min crosslink under codes PDB-ID 9C8U and EMD-43625 , and PRC2, 10 min crosslink under code EMD-45273 [ https://www.ebi.ac.uk/emdb/EMD-45723 ]. Raw datasets (movies) and their respective gain references were deposited in the Electron Microscopy Public Image Archive (EMPIAR): PP–AavLEA1, 1:8 molar ratio under accession code EMPIAR-11963 , PP–AavLEA1, 1:40 molar ratio under accession code EMPIAR-11964 , PP–RvLEAM short , 1:6 molar ratio under accession code EMPIAR-11965 , PP–CHAPSO 4 mM under accession code EMPIAR-11966 , PRC2–AavLEA1, 1:40 molar ratio under accession code EMPIAR-11975 , PRC2–RvLEAM short , 1:6 molar ratio under accession code EMPIAR-11976 , PRC2–RvLEAM short , 1:6 molar ratio, 2 min crosslink under accession code EMPIAR-11978 , PRC2–RvLEAM short , 1:6 molar ratio, 10 min crosslink under accession code EMPIAR-11979 , and PRC2, 10 min crosslink under accession code EMPIAR-12125 . The PRC2–RvLEAM short , 1:6 molar ratio with 10 mM MgCl 2 dataset is uploaded as aligned micrographs, under accession code EMPIAR-12140 . Reconstructed tomograms for PP–AavLEA1, 1:8 molar ratio are included in EMD-43619 and PRC2–RvLEAM short , 1:6 molar ratio in EMD-43621 . The following previously published PDB depositions were used in this study: PDB-ID 8FYH and PDB-ID 5EXR .  Source data are provided with this paper.

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Acknowledgements

We are grateful to Tom Cech, Anne Gooding, and Jiarui Song at the University of Colorado Boulder for their generous gift of purified recombinant human PRC2. We also extend our thanks to members of the Lim and Grant laboratories for their valuable suggestions. Our appreciation goes to Kliment Verba and his lab members at the University of California San Francisco for their assistance with our ppSSNR calculations. We thank Lori Passmore and her lab members at MRC LMB for providing insightful feedback on our preprint. We also like to thank Tom Terwilliger at the Los Alamos National Lab for his help in model refinement using the Phenix software. Lastly, we are thankful to our colleagues Elizabeth Wright and Robert Kirchdoerfer for their helpful feedback and suggestions. Some of this work was performed in the Cryo-EM Research Center (CEMRC) in the Department of Biochemistry at the University of Wisconsin–Madison. We thank the staff at CEMRC for their support and assistance in cryo-EM data collection. The Lim lab is a member of the SBGrid consortium ( www.sbgrid.org ) and some of the analyses were performed using software compiled by SBGrid. Support for this research was provided to C.L. by the National Institutes of Health (NIH), the National Institute of General Medical Sciences (R00GM131023 and DP2GM150023), and the University of Wisconsin–Madison, Office of the Vice-Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation and the Department of Biochemistry. In addition, K.M.A. is supported by an NIH T32 predoctoral fellowship (T32GM130550). T.G. is an Investigator of The Morgridge Institute for Research.

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Kaitlyn M. Abe, Gan Li, Qixiang He, Timothy Grant & Ci Ji Lim

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Contributions

C.L. conceived the study. K.M.A. and Q.H. made the recombinant proteins. K.M.A made the cryo-EM grids, and performed data collection and image processing with C.L. in support. C.L. and K.M.A. collected the cryo-EM tilt-series datasets with T.G. in support. G.L. and T.G. reconstructed the cryo-EM tomograms and performed related analyses. K.M.A. performed the crosslinking experiments and analysis. K.M.A., G.L., T.G., and C.L. wrote the manuscript and prepared the figures.

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Correspondence to Ci Ji Lim .

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A provisional patent has been filed by C.L. for this technology with the Wisconsin Alumni Research Foundation (WARF). The remaining authors declare no competing interests.

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Abe, K.M., Li, G., He, Q. et al. Small LEA proteins mitigate air-water interface damage to fragile cryo-EM samples during plunge freezing. Nat Commun 15 , 7705 (2024). https://doi.org/10.1038/s41467-024-52091-1

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DOI : https://doi.org/10.1038/s41467-024-52091-1

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