Cryo–electron microscopy breaks the atomic resolution barrier

https://www.sciencemag.org/news/2020/10/cryo-electron-microscopy-breaks-atomic-resolution-barrier-last

by nathandaly • 6 years ago

377 91 6 years ago

nathandaly• 6 years ago

FYI - This means they're able to image individual atoms, not that they're imagining WITHIN atoms as the title somewhat implies.

That is: atomic resolution, not sub-atomic resolution

stanrivers• 6 years ago

This is helpful context. I am not a scientist in any way, shape or form, but this is cool stuff. Appreciate the translation to layman’s terms!

natechols15• 6 years ago

Pedantic note: they're not really imaging individual atoms directly; it's a composite view of the structure based on averaging thousands of different images of copies of the molecule in different orientations. This is true of other structure determination methods such as crystallography of course. The breakthrough here is that these are exceptionally accurate measurements; when I tried learning EM in the early 2000s it was still mostly just vague blobs.

steve_adams_86• 6 years ago

I remember when those vague blobs were a monumental achievement. It’s very cool to see these kinds of technologies break through and get refined like this.

MayeulC• 6 years ago

I don't do much experimentation, but I am pretty sure that you can very easily distinguish individual atoms with electron beam microscopy, and get pretty close using atomic force microscopy. And I am not sure what exactly is the resolving power of synchrotron radiation, but I expect if to be pretty high.

However, the breakthrough here seems being able to do so using a low-energy electron beam. All the methods I quoted above are probably impractical or damaging with biological samples.

__MatrixMan__• 6 years ago

Pedantic note: At this scale, a particle is nothing more than a distribution of the probability of making some kind of measurement. So composites of multiple samples aren't a suboptimal means of imaging atomic structure, they're the only way. To do anything else would be to show only part of the probability field that is the atom.

(Right? I've been going back and forth about this since I saw those "videos" in 2015 of "individual photons". My gut reaction was that those aren't videos, they're simulations that cleverly represent the probability of measuring a photon as the brightness of of the "photon", but since we're measuring inherently probabilistic things maybe that's the only honest way to do it, in which case perhaps we ought to accept them as images.)

dekhn7• 6 years ago

at around 0.7A resolution, you start to be able to see electron density between atoms. Nobody using these methods cares about intranuclear structure, just the electronic structure around the nucleus.

avz• 6 years ago

Indeed. For comparison: atoms are about four orders of magnitude larger than atomic nuclei. The size of the former is generally a few tens to low hundreds of picometers (1 pm = 1e-12 m) while the size of the latter is just a few femtometers (1 fm = 1e-15 m).

See, e.g. https://en.wikipedia.org/wiki/Atomic_nucleus for concrete example sizes.

hovden• 6 years ago

The electron wavefunction scatters primarily off the nucleus potential. The electron cloud acts to screen the potential, but this effect is weak and not easily detected in the scattered wave.

edna3143• 6 years ago

Naive question: these images are made at low but finite temperatures, so each atom would fluctuate within its position such that there should be an uncertainty. Is that uncertainty lower than 1 angstrom? Otherwise, how can one talk about atomic resolution if fluctuations are larger?

natechols15• 6 years ago

The uncertainty also comes from averaging many images together. But yes, it's less than 1 Angstrom; the deposited structures (for example https://www.rcsb.org/structure/7A6A) record atomic uncertainties, "B-factors" or "temperature factors" in crystallographer-speak. In the example I pulled out the B-factors are in the low-double digits for the main-chain atoms, and if I'm remembering the math correctly, this will correspond to atomic displacements with a radius around 0.5 Angstrom. (Of course that's just an estimate by the software that performed the model optimization, but my understanding is that this part is relatively straightforward.)

_Microft• 6 years ago

Since people might not be familiar with the unit Angstrom used in the article: it is a shorthand for 10^-10 m = 0.1 nm = 1 Å (Ångström).

Edit: Wikipedia entry: https://en.wikipedia.org/wiki/Angstrom

vikramkr• 6 years ago

Cryo-EM is such a powerful and important tool in biochemistry that any advancement is bound to have significant impacts. Its difficult to overstate how powerful a tool this is and how widely used it is to understand protein structures. Essentially, it let's you see what proteins look like, with some significant advantages over previous techniques like xray crystallography for many use cases. The technique won the 2017 chemistry nobel and was method of the year for nature in 2016, even without atomic resolution.

https://www.nature.com/articles/nmeth.4115

dekhn7• 6 years ago

It's powerful, and a definite step up over crystallography in some ways, but personally I think structural biology has repeatedly been oversold as a method to understand the function of proteins at the molecular level. I say that as somebody who obtained a PhD in NMR spectroscopy and worked for 15 years on structural biology problems.

I've come to see SB as a visualization tool that helps form hypotheses, but not an actual method by which most protein structural/function problems can be directly solved.

The techniques which seem to provide the most utility are fluorescent imaging with light microscopy, often at low magnification. These are more amenable to scale-up and machine learning, both of which are likely to be necessary to unlock the next level of understanding biology.

natechols15• 6 years ago

I think your criticism of structural biology can be applied to any methods-focused investigation, as opposed to biology-focused. I've certainly been down the "okay we have the data, what does it mean?" path more times than I care to remember. Unfortunately I think this is a natural consequence of working on cutting-edge methods - structural biology has traditionally required a degree of over-specialization.

darkerside• 6 years ago

I've come to see SB as a visualization tool that helps form hypotheses, but not an actual method by which most protein structural/function problems can be directly solved.

This seems to understate the challenge and power of generating good hypotheses.

DrScientist2• 6 years ago

Exactly.

There are obviously famous exceptions - like the structure of DNA revealing key functional aspects.

But for the most part it's like claiming photography is the key to understanding how animals really behave.

Single pictures in isolation are typically not very helpful in understand the biology.

Even the promise of having a structure and therefore being able to calculate physio-chemical properties has been much harder and slower than the hype.

jcims2• 6 years ago

I'm just an interested layman but it seems like this technology might be useful in developing therapies that either suppress or amplify immune response (e.g. diabetes, vaccines, cancer). For example, it may be possible to optimize the targeting of PD-1 inhibitors by comparing the PD-L1 ligands of healthy and diseased tissues in a cancer patient (edit: found something [2]). There was also a story [1] about how more advanced imaging was used to help identify antibodies that would be effective against SARS-CoV-2.

[1]

https://www.diagnosticimaging.com/view/role-emerges-imaging-...

https://www.nih.gov/news-events/nih-research-matters/novel-c...

[2]

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050107/

mensetmanusman2• 6 years ago

We need to know the elemental sequence of every human protein at some point, so we might as well start now.

Deepmind has made some interesting progress on modeling molecular structure from first principles using advanced ML architectures. Hopefully years from now these types of tools will let us understand the in vivo structural dynamics of these amazing molecular machines that give rise to life.

hobofan4• 6 years ago

Doesn't your disclaimer kind of also explain a bias against SB, as NMR is mostly used for protein dynamics elucidation (vs. the more rigid structures of Cryo-EM)? Or do you actually mean all fields of SB?

letitgo12345• 6 years ago

It's a bit trickier than that as the flash freezing process is on the scale of milliseconds iirc which can affect what ensemble of folds are seen via cryo-em

Santosh83• 6 years ago

So at what point do we hit the Uncertainty Principle of disturbing atoms so strongly that we're no longer sure of their velocity or position?

jdmichal• 6 years ago

Do you mean the uncertainty principle or the observer effect? From Wikipedia:

https://en.wikipedia.org/wiki/Uncertainty_principle

In quantum mechanics, the uncertainty principle (also known as Heisenberg's uncertainty principle) is any of a variety of mathematical inequalities asserting a fundamental limit to the precision with which the values for certain pairs of physical quantities of a particle ... can be predicted from initial conditions.
Historically, the uncertainty principle has been confused with a related effect in physics, called the observer effect, which notes that measurements of certain systems cannot be made without affecting the system, that is, without changing something in a system.

The former just means that the more precisely we know the location, the less precisely we can predict the momentum given the initial information of the system. It has nothing to do with "disturbing the atoms"; as Wikipedia explains it's a property of wave-like systems.

https://en.wikipedia.org/wiki/Observer_effect_(physics)

On the other hand, the observer effect simply says that measurements involve active processes and those can effect the system. But it has nothing to do with velocity or position per se, just the fact that measurements can have effects.

tgbugs3• 6 years ago

UP doesn't really apply here. The data the underlies the reconstructions requires massive averaging of individual objects as part of the tomography (see Radon transform for the core approach). In addition those objects/particles need to have the requisite symmetry/similarity/regularity in structure (not universally applicable), or the population of them needs to be completely homogeneous (hard to achieve). I suspect that there are now better techniques for detecting outliers so homogeneity might not be as important.

bodhiandpysics1• 6 years ago

So in some sense that's what this is all about. The resolution of an electron microsocope is dependant on the energy of the electron, but higher energy electrons rip your sample apart. The trick that cryo-em does is to stiffen the structure of the protein by embedding it in a glass matrix (the glass is actually just really cold water that is prevented from freezing). Since the protein is stiff, you can use higher energy electrons, and thus get a higher resolution.

cinquemb• 6 years ago

Are things besides water used? Like a vacuum or something else? Would the stiffness be dependent on the energy density gradient between what it is embedded in?

The technique reminds of those i've seen in sparse eigen decomposition of a matrix, where some non square matrix X is embedded in a larger sparse matrix of zeros of the form:

⎡ zeros(X.n_rows, X.n_rows) X ⎤

⎣ X.t() zeros(X.n_cols, X.n_cols) ⎦

Except for in this case the larger matrix of zeros would be replaced by a repeated structure. Though I can't help but think that using non zeros would get some weird energy feedbacks (due to the interaction of the protein with the embedding matrix molecules) that would require some renormalizations.

alextheparrot• 6 years ago

Interesting to see the work being done to try to move structural biology towards a place where we can observe biological processes more easily. Crystal structures (Especially with ligands) were really useful to determine structure and function, but you sometimes get artifacts and it is hard to see exactly how things transition (Assuming you even get it to crystallized in the first place).

Techniques like cryo-EM and smFRET are definitely helping bridge this toolset gap to do better functional analysis of proteins and complexes. Definitely worth the Nobel Prize it won a few years back.

natechols15• 6 years ago

You can still get artifacts due to freezing. But the lack of crystal packing makes it much easier to detect functional conformational changes in different copies of the molecule on an EM grid.

fabian2k• 6 years ago

The progress of Cryo EM has been extremely impressive. I wouldn't have thought you could push it to actual atomic resolution. Even with X-Ray you don't achieve that for typical protein structures.

It'll be interesting to see if and how quickly these advances can be put into routine use.

sp332• 6 years ago

Next, researchers will strive to achieve similar sharp resolution with less rigid, large protein complexes, such as the spliceosome

Does this mean we don't currently have a good picture of what the spliceosome looks like? That seems important!

Nth_step• 6 years ago

We do. For the entire splicing cycle, via cryoEM:

"Molecular Mechanisms of Splicing: Spliceosome Machinery and Pre-Messenger RNA Splicing Cycle"

https://youtube.com/watch?v=OuAGeQYjfus

natechols15• 6 years ago

Most of these still count as low-resolution - I don't see anything better than 3 Angstrom in the PDB, most are much worse. More than good enough for movies and functional assays, but some of the fine details will be guesses.

martinpw• 6 years ago

What does the color information represent in the image?

flobosg4• 6 years ago

Such color schemes usually represent the position of the amino acid in the protein sequence, from blue (start, or N-terminus) to red (end, or C-terminus). Keep in mind that the figure shows an oligomer of apoferritin, formed by several individual protein chains.

natechols15• 6 years ago

It's also possible it's colored by uncertainty in the atomic positions - what crystallographers call the "B-factor".

aendruk• 6 years ago

Looks like it's just distance from the center, probably for the sake of visualizing 3D space in a press release.

flobosg4• 6 years ago

That's another plausible option that I forgot to consider.

sradman• 6 years ago

The paper Atomic-resolution protein structure determination by cryo-EM [1]:

Single-particle electron cryo-microscopy (cryo-EM) is a powerful method for solving the three-dimensional structures of biological macromolecules.
At resolutions better than 4 Å, atomic model building starts to become possible, but the direct visualization of true atomic positions in protein structure determination requires much higher (better than 1.5 Å) resolution, which so far has not been attained by cryo-EM.
Here we report a 1.25 Å-resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope that provides, to our knowledge, unprecedented structural detail.

[1] https://www.nature.com/articles/s41586-020-2833-4

flobosg4• 6 years ago

There are actually two papers being cited. The other one is titled Single-particle cryo-EM at atomic resolution: https://www.nature.com/articles/s41586-020-2829-0

irjustin• 6 years ago

Can I just say how amazingly well articulated this article was?

It was so clear, lacked fluff and explained enough of an extremely complex area of science to have basic grasp of the limitations, difficulties and gains that were made in the latest advancement.

I really wish more articles would be like this.

tgbugs3• 6 years ago

Some readers here might be interested in the history of the technology that has enabled cryo-EM. One of the key innovations was the development of CMOS direct detection devices that are able to detect electrons with extremely high signal to noise ratio, operating very close to the thermal limit of the DDD itself. I think it is an excellent example of the fundamental interdependence of cutting edge research and cutting edge engineering. Some (maybe most?) of the initial research and development [0, 1] on the cameras was done in my grandadvisor's lab. Another part of the story is related to the massive improvements in algorithms that in some cases have enabled nearly real time reconstruction and visualization of viruses from cryo-EM samples (I don't have links for these handy), and the work to increase the resolution of the reconstructions presented in the linked article.

0. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2359769/ 1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210420/

rhn_mk1• 6 years ago

What's "DDD"?

tgbugs3• 6 years ago

Direct Detection Device.

DoofusOfDeath3• 6 years ago

Layman's question: if we know the precise shapes of two molecules, is that enough information for us to compute every interesting detail about how they'll interact?

radus• 6 years ago

No, not by a long shot unfortunately.

hobofan4• 6 years ago

Especially for proteins there is not a single "precise shape" for the whole protein. It might take on slightly different shapes dependent on a number of factors (e.g. pH of the environment). Interactions between a protein and some other molecule might also alter the shape of the protein. So overall, a slight change in any of those values can have cascading effects in how it interacts with any other molecule (which themselves share the same problem).

DoofusOfDeath3• 6 years ago

So you're saying that given a particular instance of a protein molecule, the surrounding environment can cause its shape to change, but the set of bonds stays constant?

Or are you saying that the shape change involves a change to the set of bonds, but for some reason we continue to call it the same "kind" of protein?

COGlory• 6 years ago

No, because presumably they're solvated and that will change things dramatically. We have no idea, for instant, what the dielectric constant of water is in the middle of a protein, and therefore, no way to do these calculations.

georgewfraser• 6 years ago

When I was in grad school I was a TA for a biochemistry class and the prof asked me to deliver the lecture on crystallography while he was on vaca. Part of the lecture was about how electron microscopy was easier but pretty inaccurate compared to x-ray crystallography. But the slides were 10 years out of date, the accuracy of EM was still not as good but the gap was much smaller. So I updated them with more recent EM results from the same labs.

When the prof came back he spent the first 5m of the next lecture "correcting" me, explaining that EM sucked, x-ray was the only good technique. I realized that he had basically made up his mind decades earlier and no matter what changed in the technology he wasn't going to change his opinion. Science has its own politics, similar to actual politics in that conclusions drive observations, but instead of being about minimum wage or housing supply it's about arcane scientific techniques.

pierrefermat1• 6 years ago

Wondering if we should go with some straight name and shaming so as to any future students can avoid things like this, be a real waste of PhD students to follow him and go into areas without any prospects

throwaway67853• 6 years ago

This is quite typical in academia, especially in biology and medicine.

I finished my PhD recently. Examiners asked me to rewrite some sections and resubmit my thesis because one of them didn't "believe" my results. Believing in this context actually meant I made lots of his stuff outdated / irrelevant using mathematical models instead of hand waving. Models led to precise findings, which had some lab validation by the time I submitted.

At the same time my university, which is a really famous one, was encouraging me to patent my results. And my supervisor applied for massive funding as if all was his idea. 2 major clinical trials are now recruiting to test my findings.

Least to say, I refused and got a new exam with some honest people.

natechols15• 6 years ago

Academic scientists can be intensely conservative, even reactionary, when confronted with new ideas and methods. If you want to see something really horrific, try digging up some of the original debate on whether experimental data deposition should be mandatory for structural biology publications. Here is the one bit I know that's online still (PDF is free): https://www.nature.com/articles/nsb0698-407

The fact that there had to be a debate on this issue at all tells you everything. The forces of good triumphed in the end but it probably took a full generation for everyone to get in line. (I've heard legends of computer-vision software being written just to decode the figures in protein structure publications from competitors. Would love to know if this is actually true, but again, the fact that it even sounds plausible tells you a lot about the broken incentives.)

zylent• 6 years ago

This is true - I work in a related field.

intricatedetail• 6 years ago

This is a some sort of scream for money. These "scientists" do it on purpose in hope that one day a new tech company will pay them millions to change their mind. These "scientists" should be rooted out.

roughly• 6 years ago

"Science advances one funeral at a time" - Max Planck

mmmBacon• 6 years ago

Sort of off-topic but I recall the first time I ran a scan on an atomic force microscope back in the early 90’s. I was totally blown away by the ability to “see” individual atoms.

The whole system was so high tech for its day; including the computer hardware which I think was a Sun workstation. It even had magneto optical drives for storing images and a high resolution color printer for printing images out. Given that I had only used DOS up to this time, it felt like to I had been teleported into the future.

Ice_cream_suit• 6 years ago

The original paper: https://www.nature.com/articles/s41586-020-2829-0

Published: 21 October 2020

Single-particle cryo-EM at atomic resolution

Takanori Nakane, Abhay Kotecha, Andrija Sente, Greg McMullan, Simonas Masiulis, Patricia M. G. E. Brown, Ioana T. Grigoras, Lina Malinauskaite, Tomas Malinauskas, Jonas Miehling, Tomasz Uchański, Lingbo Yu, Dimple Karia, Evgeniya V. Pechnikova, Erwin de Jong, Jeroen Keizer, Maarten Bischoff, Jamie McCormack, Peter Tiemeijer, Steven W. Hardwick, Dimitri Y. Chirgadze, Garib Murshudov, A. Radu Aricescu & Sjors H. W. Scheres

Nature (2020)

Cryo–electron microscopy breaks the atomic resolution barrier

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