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I'm a structural biologist that uses CryoEM to study archeal viruses. I'd be happy to answer any questions about CryoEM. I did want to point out though that th

I'm a structural biologist that uses CryoEM to study archeal viruses. I'd be happy to answer any questions about CryoEM. I did want to point out though that this title is misleading. The researchers didn't get an image of an atom. Instead, they reconstructed hundreds of thousands of images of the protein to determine where the atoms are almost exactly. So there's no actual image that shows an atom, as cool as that would be.

mynegation 6y ago
What are the limitations of this technique in determining spatial structure of the proteins? Do they get denaturated when cooled?
- gewa 6y ago
  No, the proteins are vitrified very rapidly in liquid ethane at approx. -200°C, so that the structure ist conserved.
  - candiodari 6y ago
    "Vitrification" is a way to freeze water so that it forms a glassy solid instead of ice crystals, locking the proteins in place and so that the "ice" reflects electrons randomly like water does, and so the only large structure redirecting electrons is the protein particles.
  - tynpeddler 6y ago
    Protein "structure" is a slippery concept. In situ measurements, molecular dynamics simulations and basic common sense pretty conclusively show that most proteins actually have a number of conformations they access during normal biological processes. In many cases, these conformations are required for the proteins normal functions. I used to work in the de novo protein field and it was a constant source of irritation to me that what we mainly designed were crystal structures, not dynamic functional proteins.
    - COGlory 6y ago
      There's lots of structural information such as conserved folds that can be gleaned from Cryo and X-ray structures. Protein dynamics is an important part but structures published with these mechanisms have been proven biologically relevant by site directed mutagenesis an almost infinite number of times now.
    - mynegation 6y ago
      Thank you. Would it be fair to say that crystallized protein is always in one specific confirmation? Compared to that, would vitrification have the chance (however random) to capture protein molecules in different conformations?
      - COGlory 6y ago
        Yes, a crystallized protein will always be in a single conformation, or you won't be able to see it because the electron density map will be an average of all possible conformations, and therefore meaningless. Single particle gives you the opportunity to see different conformations, but only if the data is discrete. If there's a continuous amount of conformations (think a molecular motor that's rotating) you would need nearly infinite data to resolve a nearly infinite number of conformations. If the data is less than continuous, you can image enough particles to see all the different conformations by constructing multiple models in parallel and using 3D angular searching to bin them by what conformation they are in. This is a computationally exhausting process, however.
        natechols 6y ago
        I hate to be that guy, but your first statement is technically incorrect: you can have discrete alternate conformations superimposed in the electron density (usually 2-3 is the most that can be resolved), and you can have different conformations of multiple copies of the molecule. (I've personally worked with both, although the differences in the second case were small.) That's not even counting ensemble-based approaches to modeling the crystal structure, although that's arguably just a different way of representing the uncertainty. All that aside, crystallization certainly biases it towards specific conformations, which single-particle EM does not.
- COGlory 6y ago
  For a long time, it was the cameras. We couldn't get smaller things into focus and still preserve high-resolution images. Now that the camera problem has been solved with CMOS detectors, the next problem is likely going to be the contrast-transfer function [1]. If we can solve that (such as with an energy filter, as I suspect the authors of this paper used), we can get smaller and smaller samples in focus without losing high resolution information from the electron scattering. [1] https://www.youtube.com/watch?v=mPynoF2j6zc https://www.youtube.com/watch?v=mPynoF2j6zc
petermcneeley 6y ago
Are there any observations that are surprising with respect to the predictions from quantum chemistry? (More specifically computational chemistry)
- COGlory 6y ago
  I'm not aware of any, but I don't think that until we're able to see proteins moving we'll actually be able to answer those questions visually. We've got a long way to go before that happens.
Ididntdothis 6y ago
How long does something have to sit still to take a picture? Is there an exposure time?
- COGlory 6y ago
  The samples themselves are plunge frozen into vitreous ice using liquid ethane, so they won't move much on their own. The electron beam, as well as the stage they sit on inside the microscope do cause movement of the grid/sample, however, and so on modern microscopes, what we actually record are movies so that we can correct for things like beam induced motion with software. Exposure is a separate variable because it also causes sample degradation. Ideally, a microscope is only shooting one electron at a time at the sample (that never happens obviously), but the fewer electrons, the better. On overexposed micrographs, you can see the burn marks on the sample.
kbradero 6y ago
what is the typical career/study path for this field ?
- COGlory 6y ago
  I did my undergraduate degree in Nutritional Science, realized I liked enzymes/protein structure a lot, and applied to a biochemistry department PhD program. I'm in my 3rd year and currently on/ahead of track so not having a biophysics background hasn't held me back. I'd recommend, if that's what you wanted to do, to start with biophysics or molecular biology as those are the hardest aspects. A good computer science background is helpful as well, because a huge part is processing terabytes upon terabytes of micrographs into a finished, ab initio protein model. There's always room for new techniques there. Personally, if I were to do it over, I'd major in the closest thing to biophysics/structural biology I could, and spend as much time in a lab doing undergraduate molecular biology research as I could.
perl4ever 6y ago
"The researchers didn't get an image of an atom" You could say the same about any image produced by a scientific instrument these days. Any astronomical image has a huge amount of data behind it, but we don't sit around saying "gee, there are no actual images, isn't that a shame". If you don't want to call something an image, you can call it a diagram produced by a data processing program, but whatever you call it, it's disappointing if it's not provided.
dang 6y ago
Ok, since at least two of you who know what you're talking about have objected to the title, I've replaced it with the more general subtitle above. Thanks! (Submitted title was "Cryo-electron microscopy technique sees individual atoms for first time".)
mensetmanusman 6y ago
Can you use CryoEM to see the atomic bonding arrangement in any material you throw at it? E.g. visualize a glass or polymer network?

mynegation 6y ago

What are the limitations of this technique in determining spatial structure of the proteins? Do they get denaturated when cooled?

gewa 6y ago
No, the proteins are vitrified very rapidly in liquid ethane at approx. -200°C, so that the structure ist conserved.
- candiodari 6y ago
  "Vitrification" is a way to freeze water so that it forms a glassy solid instead of ice crystals, locking the proteins in place and so that the "ice" reflects electrons randomly like water does, and so the only large structure redirecting electrons is the protein particles.
- tynpeddler 6y ago
  Protein "structure" is a slippery concept. In situ measurements, molecular dynamics simulations and basic common sense pretty conclusively show that most proteins actually have a number of conformations they access during normal biological processes. In many cases, these conformations are required for the proteins normal functions. I used to work in the de novo protein field and it was a constant source of irritation to me that what we mainly designed were crystal structures, not dynamic functional proteins.
  - COGlory 6y ago
    There's lots of structural information such as conserved folds that can be gleaned from Cryo and X-ray structures. Protein dynamics is an important part but structures published with these mechanisms have been proven biologically relevant by site directed mutagenesis an almost infinite number of times now.
  - mynegation 6y ago
    Thank you. Would it be fair to say that crystallized protein is always in one specific confirmation? Compared to that, would vitrification have the chance (however random) to capture protein molecules in different conformations?
    - COGlory 6y ago
      Yes, a crystallized protein will always be in a single conformation, or you won't be able to see it because the electron density map will be an average of all possible conformations, and therefore meaningless. Single particle gives you the opportunity to see different conformations, but only if the data is discrete. If there's a continuous amount of conformations (think a molecular motor that's rotating) you would need nearly infinite data to resolve a nearly infinite number of conformations. If the data is less than continuous, you can image enough particles to see all the different conformations by constructing multiple models in parallel and using 3D angular searching to bin them by what conformation they are in. This is a computationally exhausting process, however.
      - natechols 6y ago
        I hate to be that guy, but your first statement is technically incorrect: you can have discrete alternate conformations superimposed in the electron density (usually 2-3 is the most that can be resolved), and you can have different conformations of multiple copies of the molecule. (I've personally worked with both, although the differences in the second case were small.) That's not even counting ensemble-based approaches to modeling the crystal structure, although that's arguably just a different way of representing the uncertainty. All that aside, crystallization certainly biases it towards specific conformations, which single-particle EM does not.
COGlory 6y ago
For a long time, it was the cameras. We couldn't get smaller things into focus and still preserve high-resolution images. Now that the camera problem has been solved with CMOS detectors, the next problem is likely going to be the contrast-transfer function [1]. If we can solve that (such as with an energy filter, as I suspect the authors of this paper used), we can get smaller and smaller samples in focus without losing high resolution information from the electron scattering. [1] https://www.youtube.com/watch?v=mPynoF2j6zc https://www.youtube.com/watch?v=mPynoF2j6zc

petermcneeley 6y ago

Are there any observations that are surprising with respect to the predictions from quantum chemistry? (More specifically computational chemistry)

COGlory 6y ago
I'm not aware of any, but I don't think that until we're able to see proteins moving we'll actually be able to answer those questions visually. We've got a long way to go before that happens.

Ididntdothis 6y ago

How long does something have to sit still to take a picture? Is there an exposure time?

COGlory 6y ago
The samples themselves are plunge frozen into vitreous ice using liquid ethane, so they won't move much on their own. The electron beam, as well as the stage they sit on inside the microscope do cause movement of the grid/sample, however, and so on modern microscopes, what we actually record are movies so that we can correct for things like beam induced motion with software. Exposure is a separate variable because it also causes sample degradation. Ideally, a microscope is only shooting one electron at a time at the sample (that never happens obviously), but the fewer electrons, the better. On overexposed micrographs, you can see the burn marks on the sample.

kbradero 6y ago

what is the typical career/study path for this field ?

COGlory 6y ago
I did my undergraduate degree in Nutritional Science, realized I liked enzymes/protein structure a lot, and applied to a biochemistry department PhD program. I'm in my 3rd year and currently on/ahead of track so not having a biophysics background hasn't held me back. I'd recommend, if that's what you wanted to do, to start with biophysics or molecular biology as those are the hardest aspects. A good computer science background is helpful as well, because a huge part is processing terabytes upon terabytes of micrographs into a finished, ab initio protein model. There's always room for new techniques there. Personally, if I were to do it over, I'd major in the closest thing to biophysics/structural biology I could, and spend as much time in a lab doing undergraduate molecular biology research as I could.

perl4ever 6y ago

"The researchers didn't get an image of an atom" You could say the same about any image produced by a scientific instrument these days. Any astronomical image has a huge amount of data behind it, but we don't sit around saying "gee, there are no actual images, isn't that a shame". If you don't want to call something an image, you can call it a diagram produced by a data processing program, but whatever you call it, it's disappointing if it's not provided.

dang 6y ago

Ok, since at least two of you who know what you're talking about have objected to the title, I've replaced it with the more general subtitle above. Thanks! (Submitted title was "Cryo-electron microscopy technique sees individual atoms for first time".)

mensetmanusman 6y ago

Can you use CryoEM to see the atomic bonding arrangement in any material you throw at it? E.g. visualize a glass or polymer network?