The Olympic pictogram chart reminded me of a 2-D class averages figure (more on what this is in a minute) – that, plus the gymnastics reminded me of proteins – plus the protein structures I told you about yesterday – had me thinking about cryo-Electron Microscopy (CryoEM) – the technique which was used to figure out those structures… (and I will take any excuse to do some more cartwheels…) So here’s an intro to cryo-EM and a bit of comparison to x-ray crystallography, which I’ve talked about a lot more.

Video’s new but text is a shortened version of a longer, more detailed post if you want those deets:

The basic workflow is:⠀

  • specimen preparation: sample (protein in some liquid) -> freeze it on a grid (try to get a super thin layer of vitreous ice (glass-like water) where the proteins don’t overlap in the squares of a tiny tiny mesh
  • imaging: take a LOT of images – go from hold to hole in the grid taking lots of pictures⠀
  • particle picking: determine what’s actually a particle (your protein or protein/DNA/RNA etc. complex) versus just background⠀
  • alignment and 2D classification: group together the images that look similar to get 2D averages⠀
  • 3D reconstruction: use those 2D averages to generate an initial 3D map (helps if you have a low-res negative stain or something)⠀
  • refinement: refine that initial map to get a refined 3D map⠀
  • make it even better to get your final 3D map⠀
  • stick an atomic model into it (make that sticky model thing showing where the individual carbons, oxygens, etc. – or at least the protein backbone ones) are located

And here’s a more detailed look

“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’m 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.⠀

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. ⠀

We use x-rays in x-ray crystallography, which have short wavelengths but can’t be focused so we have to do a bunch of mathematical tricks to work of off “diffraction patterns” from scattered rays instead of using a microscope. So, with cryo-EM, we instead use electron waves – an “electron gun” sends electrons 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).⠀

The freezing is usually done on a carbon-coated metal (often copper or gold) grid. These are really small wire meshes (kinda paper hole-punch size) – usually 3.05mm across with 200-400 squares per inch (so a “200-mesh” grid has 20 squares in each direction, each (hopefully) with a film of vitreous water with particles suspended in it. ⠀

The metal is often coated in a layer of carbon. This carbon coating is used because it’s unreactive and not very sticky – so you don’t have to worry about your molecule binding to the grid itself and only letting you see a couple angles. You don’t want your sample interacting with the grid itself at all – instead you want the particles to be suspending in the liquid films filling the grid holes (think kinda like a bunch of bubble wands). These films are really thin – a few hundred to a few thousand Å – you don’t want too thick because you only want a single particle per layer and you also don’t want a bunch of background signal. To get that thinness, you can use a Vitrobot machine that reminds me of one of those monkey cymbals things  – it smooshes the grid with filter paper to remove excess. ⠀

So now you have your sample. You put it in the microscope and shoot electrons at it. Then what? Some of the electron waves undergo “elastic scattering” which is like your classic “billiard ball bouncing off a wall” example – A wave hits one thing and just bounces off – its direction gets changed a little, but it doesn’t lose any energy – and now you need to bend these jumbled waves back to hit the detector using other magnetic “lenses.”⠀In addition to just hitting the detector at slightly different places, the waves of electrons can have different amplitudes (strengths). And as a result of this, you’re able to get 3D information instead of just 2D like you’d get from a shadow of something completely solid. Instead of a shadow, these are “projection images” – you get different projection images depending on the orientation of the particle. And in the “2D classification” step, you sort the ones with similar orientations before going through all the even harder work of getting out the 3D info. ⠀

But just finding the particles is hard. You need a lot of them in order to get enough information and they’re hard to find even when they’re there! One of the main problems in cryo-EM is “contrast” – turns out biochemical molecules are pretty good at “camouflage” – they only interact weakly with the electrons, so they blend into the background (and are super tiny) so it’s hard to tell signal from noise – there’s a poor signal to noise ration (SNR). ⠀

“particle picking” – in this stage of the process, the computer (with help from the researchers) decides what is an actual particle (e.g. the protein, multi-protein complex, protein/DNA complex, etc.) versus what’s just the background. Because the signal from any one particle is so weak, it tries to pick a lot a lot of them. ⠀

Then it tries to sort them based on which look similar. Each image contains 3D info, but the computer at this point treats them as simple 2D images. So it goes through and groups the similar-looking ones together into a number of 2-D classes – usually these 2-D classes are different orientations of the molecules (e.g. top view, side view). The researcher tells the computer how many classes to make & it uses a translation-rotation function – basically it shifts and turns the picture to see how well it fits with the others in the class and finds the best match. ⠀

Then it’s on to 3D classification. The images in the 2D classes are used to build a rough “map” of the 3D protein they came from. The reason this is possible is because of something called the central projection theorem, which I’m not going to get into. ⠀

You can further refine and refine the map and get a final map and then build an atomic model into it. ⠀

Unlike in crystallography, where the highest achievable resolution is limited by the wavelength of light, since the electron waves are so much smaller, the wavelength isn’t limiting – instead it’s the pixels that provide the maximum resolution limit – the smaller the pixel, the better able to tell tiny differences in where exactly an electron hits the detector. So scientists are frequently trying to up the pixels.⠀

But, just like in crystallography, most of the time you’re limited most by the sample itself – not that it’s always the researcher’s “fault” – some molecules are just tricky to image (and it’s not their fault either – you ripped them from their natural environment, remember and now you’re complaining they’re not smiling pretty for the camera?!).⠀

Which takes us to some more differences between cryo-EM & crystallography. It’s not like you can always just randomly take your pick, or that, now that cryo-EM technology has advanced you should give up that crystallography stuff. Cryo-EM works well for big things that provide lots of contrast – so it’s great for big complexes. Which is basically the opposite of what crystallography’s good for. ⠀

The major advantage of cryo-EM is that it doesn’t require crystallization. Because crystallography requires that perfect orderliness, the fewer potential particle to particle “variables” the better – and the more parts there are the more such “variables” you have to get to organize. But, for small, well-structured particles, crystallography can provide you detail at higher-res. And it’s great for things like showing drug binding, etc. ⠀

But getting something to crystallize usually means a lot a lot of screening & optimizing and requires a lot a lot of protein. Plus, even if you do manage to get something to crystallize, sometimes the molecules adopt slightly “unnatural” positions due to crystal packing interactions.⠀

But it’s not like getting good cryo-EM data is a walk in the park – it takes a lot of optimization too – as I’ve seen my colleagues go through!⠀

more on vitrification:

more on x-ray crystallography: 

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