Cryo-electron microscopy breaks a key barrier • Hacker News Board

Re: Cryo-electron microscopy breaks a key barrier

#71

Post by thereisnospork » Thu, Jun 04, 2020, 8:26 PM UTC

twic wrote: post #26

TomJansen wrote: post #18

Earlier quoted context omitted.

Oh please, this philosophical stuff is unnecessary. Cyro-EM is about a decade old and is just an alternative to X-Ray Crystallography for elucidating the structures of proteins. This article describes a way to improve the resolution of Cyro-EM, matching it to X-Ray Crystallography. The biggest bottleneck of X-Ray Crystallography is the production of protein crystals which is not necessary with Cyro-EM.

It's unbelievable how cryo-EM has come on. When i was a student, it was an also-ran behind crystallography and NMR, something that a few weird groups used to study a few special cases. Now you have to wonder if there's any point doing crystallography at all! I still like NMR, because you're looking at proteins in solution. But if the freezing is good in cryo-EM, maybe even that becomes moot.

I'm not sure if it applies to proteins, but for smaller molecules there seemed to be some really interesting results a few years ago about using MOF's to adsorb analytes in a spatially repeating manner so x-ray diffraction techniques could be used without crystallization. Haven't followed closely enough to know if anything is panning out though.

Re: Cryo-electron microscopy breaks a key barrier

#72

Post by COGlory » Thu, Jun 04, 2020, 8:27 PM UTC

rezna wrote: post #60

COGlory wrote: post #36

Earlier quoted context omitted.

There aren't any. The way cryo-EM works is it images hundreds of thousands of copies of the molecule and uses those to construct a 3D model, in this case, with atomic resolution. However, a single micrograph doesn't necessarily contain atomic resolution.

No longer need for hundreds of thousands. The aim is for just few thousands in few hours to reach acceptable resolution. The development goes real fast with both SW and HW.

Depends greatly on whether or not what you're imaging has symmetry.

Re: Cryo-electron microscopy breaks a key barrier

#73

Post by COGlory » Thu, Jun 04, 2020, 8:29 PM UTC

tynpeddler wrote: post #64

gewa wrote: post #43

Earlier quoted context omitted.

No, the proteins are vitrified very rapidly in liquid ethane at approx. -200°C, so that the structure ist conserved.

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…

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.

Re: Cryo-electron microscopy breaks a key barrier

#74

Post by mynegation » Thu, Jun 04, 2020, 8:30 PM UTC

tynpeddler wrote: post #64

gewa wrote: post #43

Earlier quoted context omitted.

No, the proteins are vitrified very rapidly in liquid ethane at approx. -200°C, so that the structure ist conserved.

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…

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?

Re: Cryo-electron microscopy breaks a key barrier

#75

Post by COGlory » Thu, Jun 04, 2020, 8:32 PM UTC

Ididntdothis wrote: post #62

COGlory wrote: post #39

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 wo…

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

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.

Re: Cryo-electron microscopy breaks a key barrier

#76

Post by jgehrcke » Thu, Jun 04, 2020, 8:34 PM UTC

While this is a breakthrough within the field of cryo-electron microscopy it is important to appreciate that for many questions in structural biology we also need to understand how protein structure changes over time.

With the presented method the structure sampling time seems to be O(10 s) which easily is about 10 orders of magnitudes slower than the dynamics we're interested in seeing.

A direct consequence of this is that for achieving atomic spatial resolution they needed to use a "rock-solid" protein -- one that has an exceptionally stiff structure (one that does not wiggle a lot). The presented method is great and cool, but this is a pretty severe limitation. Most proteins wiggle a lot :-).

Background: protein structure-function relationship can often be well understood only when considering the structural dynamics of the protein (key words: conformational changes, the entropic contribution to free energy).

That is, in the ideal case we would be able to measure molecular structure not only at high spatial resolution, but also at high temporal resolution.

How can one make such a measurement much faster, by ~10 orders of magnitude? By irradiating a lot of light. Via X-ray free electron lasers (XFEL).

XFEL-based techniques are expected to revolutionize structural biology (as always, also still a long way to go):

> XFEL protein crystallography not only determines high resolution structures of proteins, but also reveals the time-stamped conformational changes of proteins.

- https://www.nature.com/articles/nmeth.3070.pdf?origin=ppub

- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678726/

(PhD in structural biology/bioinformatics, investigated dynamics of proteins with molecular dynamics simulations)

Re: Cryo-electron microscopy breaks a key barrier

#77

Post by COGlory » Thu, Jun 04, 2020, 8:35 PM UTC

kbradero wrote: post #69

COGlory wrote: post #39

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 wo…

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

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.

Re: Cryo-electron microscopy breaks a key barrier

#78

Post by COGlory » Thu, Jun 04, 2020, 8:36 PM UTC

petermcneeley wrote: post #41

COGlory wrote: post #39

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 wo…

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

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.

Re: Cryo-electron microscopy breaks a key barrier

#79

Post by natechols » Thu, Jun 04, 2020, 8:36 PM UTC

mncharity wrote: post #70

natechols wrote: post #68

Earlier quoted context omitted.

The terrain analogy is great - this is how the first structural biologists had to view electron density: https://image.slideserve.com/105608/electron-density-maps-of... I think this is a stack of 2d "slices" through the density, printed on transparencies. But I've also seen 2d representations of charge density (some of which is just theoretical studies) that are even more similar to a terrain map.

Neat. I've been exploring visual representations of electron density, optimized for educational use with XR. Intending to illustrate that science education content could be made transformatively less wretched. Isosurfaces and nonlinear color gradients are educationally problematic. And fuzz gradients are hard to visually stereo fuse. And on near-term AR displays, black is transparent, and background environment color…

I've been out of the field for a few years, but I know that the graphics hardware (and accompanying software tools) make what you're describing much easier. This is best example I can come up with: https://jcheminf.biomedcentral.com/track/pdf/10.1186/1758-29... (see page 12)

Random asides: 1. once you're used to it, the simple isosurface mesh is super easy to work with (i.e. build molecules into), and for structural biologists the broad approximation is perfectly adequate 2. even so, the amount of unexplained blobs - some of which are definitely not noise - in my (xray) electron density maps was always both fascinating and frustrating. that's one reason why we don't spend much time on fancy renderings of experimental density at anything worse than subatomic resolution, because you're just sharpening the features that your atomic model doesn't explain.

Re: Cryo-electron microscopy breaks a key barrier

#80

Post by COGlory » Thu, Jun 04, 2020, 8:38 PM UTC

mynegation wrote: post #74

tynpeddler wrote: post #64

Earlier quoted context omitted.

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…

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?

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.