X-ray crystallography and cryo-em (cryo-electron microscopy): an overview & comparison

Dying to know “What’s the deal with cryo?” Cryo-Electron Microscopy (CryoEM) has transformed the structural biology field in recent years, and even garnered some wider fame when Jacques Dubochet, Joachim Frank and Richard Henderson won the Nobel Prize for it in 2017. So what is it? How does it compare the the “traditional” x-ray crystallography? And is crystallography dead? (hint: NO!) Each of these techniques has pros and cons. So let’s take a look at what’s going on. Starting with “What is structural biology anyway?” (I didn’t really even hear this term until late in undergrad and people kinda just expected me to know and never really explained, so here’s my attempt at it…)

“Structural biology” is a sub-field of biology that’s focused on figuring out what molecules “look like” and how their shape is connected to their function. A little more on this later, but for now just think of a silver fork and spoon. They’re made up of the same material so just figuring out “hey these are made of silver” isn’t going to tell you much about what they do (but feel free to pat yourself on the back - I’m all for celebrating little wins!). If you play around with them blindly (and carefully), you might be able to figure out that you can eat soup with a spoon but not a fork but it’s easier to stab and eat pasta noodles with your fork. And if you can see them, it’s easy to see how their distinct shapes allow them to perform differently at these tasks.

Structural biology works to connect the “what it does” to the “what it looks like” to help determine “how it does it” and it uses techniques including X-ray crystallography and cryo-EM to get looks, combined with biochemical & biophysical techniques (measuring activity, binding, etc.) to further investigate the functional importance of different parts that are seen (though sometimes this functional stuff is discovered first but makes a lot more sense once you can see it). It’s kinda like how if you make a hole in the spoon-y part of the spoon it makes your soup drip before it gets to your lip & if you see the spoon shape you can predict where hole-making might make you dribble. But, if you make a hole in the handle you might not know.

And speaking of things you might not know, let’s discuss “cryo” - “cryo” (which my computer insists on de-correcting to cry, so apologies if I don’t catch all the “corrections”) is short for “cryogenic” meaning really really cold (which we need to keep the molecules safe(ish) and still(ish)). And when structural biologists say “cryo” they’re usually referring to single particle cryo-electron microscopy (cryoEM) as opposed to things like cryo-tomography, which looks at “slabs” like sections of cells or tissues.

You might sometimes hear it called single *molecule* cryoEM, but the things you’re looking at don’t have to be individual molecules. A molecule is something where all the atoms (the individual carbons, hydrogens, etc.) are connected through strong covalent bonds (like one protein chain) as opposed to “complexes” where multiple molecules can interact with one another through weaker interactions (like multiple protein subunits and RNA pieces working together to make a ribosome). So “particles” is a broader term that encompasses single proteins, multi-protein complexes, protein/DNA complexes, etc. - any sort of individual “particles” in solution (i.e. each particle surrounded by its own full water coat).

Disclaimer - even though I earned my PhD right down the hall from a top-of-the-line cryo-electron microscope, I personally don’t use cryo-EM, but have done some crystallography and will talk more about this at the end.

So imagine you have a bunch of copies of one of these particles. And you want to figure out what they look like…

If you want to look at something small, your first thought might be - let’s use a microscope. Microscopes make things look bigger by taking advantage of the wave properties of light (visible light is a form of ElectroMagnetic Radiation (EMR) - which can be thought of as packets of energy called photons traveling as waves). When a wave interacts with things, the wave’s path can get altered. So a visible microscope can shine light through something and have that thing alter the light waves’ paths. Thanks to that altering, you now you have a bunch of waves “out of phase” (out of step with one-another “peak-wise”). So the signal from your thing is kinda jumbled.

finished in comments

[thebumblingbiochemist]

Visible light microscopes then re-focus the light before you see it - but in such a way that the image looks bigger than the real thing was, so it’s easier to see it and tell different parts of it apart (resolve details). This is possible because the phenomenon of “refraction” causes visible light to bend going when it goes through different media (like going from air to glass to air). So, before you see the light, microscopes have those jumbled waves travel through glass lenses which, because of their curves & thicknesses, bend the waves back to a focal plane where the signal appears as a (bigger) image.

That works for things that are “small” in the sense most people think about small things, but not “small” at the level that structural biologists think about things. Structural biologists usually talk in terms of angstroms. An Angstrom (Å) is 10⁻¹⁰ meter, or 0.1 nanometers (nm) and the average protein has a diameter of ~5nm, so 50Å. Problem is, microscopes are limited by the wavelength of the light - waves are only useful for looking at things that on the same order of magnitude or bigger than their wavelength. The shortest-wavelength visible light is ~700 nanometers, so ~7000Å, whereas interatomic distances are closer to 1Å. So we can’t use visible light to resolve what we want to see.

This is why we use x-rays in x-ray crystallography - X-rays are “just” a more energetic form of visible light - they’re just different sections of the EMR spectrum. The only difference is that in x-rays the photons have more energy and the waves peak more often (higher frequency) and the peaks come closer together (shorter wavelength) so that, linearly, it’ll take a photon of an x-ray the same amount of time as a photon of visible light to go from one side of the room to another. X-rays have wavelengths that range from 0.01-10 nanometers (so 0.1-100Å) and for protein crystallography, we usually use x-rays with wavelengths of ~1 Å.

Great. We have waves we can use! We beam them at our sample and our sample scatters them. Now we just need to focus those scattered x-rays right? Um, there’s a problem. We can’t just focus x-rays like we could focus light! They’re too energetic and short-waved, so (even if they didn’t just fry the lens) they don’t wouldn’t refract when they went through glass - or any other material we could use. As a result, in order to get information from the scattered x-rays, we have to work with these “jumbled waves” directly (or at least evidence of where they hit a detector).

The jumbled waves interact with one another - due to the regular ordering of the crystal, most scattered waves cancel out, but some strengthen each other to produce a “diffraction pattern” - a series of spots on a detector. And these spots represent what we call “reciprocal space” - a weird place where things that were once close together are now far apart, along with other eccentricities I won’t go into here. But basically you can use a thing called a “Fourier transform” to go from reciprocal space to “real space.” But you have to know the “phases” of the waves - where they peaking or troughing when they hit? And we lose this information in crystallography so we have to use complicated tricks to try to find them.

That can be a lot of work… so, what if, instead of using x-rays, we use different type of super short wave. One that we *can* focus? Such an optical option is provided by electrons, but it comes with its own problems including tending to destroy your samples… Which is where the super-cooled-ness of cryo-EM will come in. But first, the electrons - as waves? I’m used to thinking about electrons as being one of 3 key subatomic particles - as in parts of the things we’re trying to look at?! Yes, there *are* electrons in our particle already - but they’re “in use” - and different from the electrons our scope will introduce!

All the stuff in our particle are constructed of atoms - of carbon, hydrogen, oxygen, etc. - And each atom is made up of even smaller parts - “subatomic particles.” At the core of every atom is a dense center called the atomic nucleus, and it’s home to positively-charged protons & neutral neutrons. Surrounding this nucleus is an “electron cloud” with negatively-charged electrons whizzing around. But the electrons we’re going to use in cryo-EM aren’t whizzing around a nucleus - they’ve broken free from the positive pull of protons in our “electron gun” and are instead traveling in a spiraling path through a tunnel towards our sample. And they’re wiggling a lot, which means that they have really short wavelengths, ~ 0.02 Å, so about 50-times shorter than the x-rays we use.

And, although we can’t focus electrons with conventional glass lenses, we *can* focus them with magnets because, for physic-y reasons, magnets are the “electron influencers” of the sub-atomic social media scene. Thanks to the “Lorentz force, ” magnetic fields are able to direct the movement of charged things (like the negatively-charged electrons). So you can use electromagnets as “lenses” to focus electrons onto your sample and then “re-focus” them onto the detector after they’ve passed through.

Sounds easy, right? Wrong. I’ve glossed over a lot of points.

First of all, it’s not like we have nicely ordered particles sitting still for the camera. Each particle is swimming around randomly doing its own thing. It’s really hard to look at something really tiny - let alone something really tiny that’s also moving all around. So we need to get them to cut that out. In crystallography, you don’t have this problem, because you’ve gotten them to organize into an orderly 3D arrangement called a lattice (kinda like a brick wall) (though getting them to crystallize is a whole ‘other problem…)

In cryo-EM, you still need the particles to stay still, but you don’t have to get them to organize - in fact you don’t want them to - instead, you freeze them in place, in all of their random orientations. So you get views from all angles. These are “projection images.” Yes - you have *images* now because we’re able to focus the scattered electron waves - unlike the x-ray waves which we had to work with scattered. You then go through the images, pick out the individual particles, find the ones that happened to be frozen in the same position, average them together and, by using some math I am *not* going to get into, use them to create a 3D model of it.

So how do you get them to stay still? This takes us back to the “cryo” in cryo-EM - as in really really cold. As in liquid ethane cooled by liquid nitrogen to keep it at -195°C, cold. So cold that molecules don’t have enough energy to wander around, thus they stay stuck in place. And if you cool them really really quickly, they don’t have time to organize into their favorite poses before they get stuck. So they freeze in place like the freeze dance. This goes for the molecules you’re trying to look at and for the water molecules around them. So, instead of forming ice (the crystalline solid form of water which has an orderly arrangement of water molecules), the water “freezes” into a “vitreous glass” where the molecules of water are stuck in place but randomly oriented - it’s still a solid, but it’s “amorphous” (no shape having).

[dpendletdpendlet]

This is nice. I like how you try to explain it as simple as possible, but no simpler. I'm in a quantum mechanics class that applies quantum theory to spectroscopy. It's a lot, but this video is short and sweet and has a lot of the foundational information needed. I feel as if I understand x ray crystallography and cryo em to an extent.

If I may, x ray crystallography shoots these rays at a crystal and those rays are scattered by the electron dense/rich atoms. The scattered waves from the electrons work in and out of phase with each other that projects an image on a detector. With these different interference patterns also comes some information about intensity. From what I understand, the intensities of the spots are related to the electron density map that you'd derive using Fourier synthesis. We can use diffraction to get the image and then from that the electron density, and then that's when you're trying to fit atoms in to the electron density map to figure out the structure.
However, one of the issues with x-ray crystallography is that you can't account for phase difference, or how the atoms are actually oriented in relation to each other.

For cryo em, you're shooting electrons at this frozen plate similar to glass with the proteins arranged in their natural composition. Shooting electrons at the frozen plate will result in an image on the detector as the electrons interact with the electrons and the nucleus in the plate. This will give an image and taking a lot of pictures from a bunch of different angles will result in a more defined 3D structure. With all of the various proteins in possibly different compositions, the different angles allow you to figure out the most prevalent structures for that protein. The issue with cryo em is typically contrast.

Does this sound right?

[madisonmarano]

Thank you so much for this! This video is incredibly helpful to a cellular biochemist who is not very good at physics but has to take the class for my major lol. I really appreciate the level of detail but lack of jargon language that makes everything confusing. You're awesome!

[CryoEMQueen]

You should make a video on helical structure determination with cryo-EM. It's a nightmare, makes SPA look easy. :)