12 ms·
Cryo–electron microscopy breaks the atomic resolution barrier
- nathandaly 6y agoFYI - 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 6y agoThis 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!
- natechols 6y agoPedantic 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 6y agoI 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.
- chrisbrandow 6y agoIn terms of either sample prep or data processing, how time consuming is this relative to X-ray crystallography? Setting aside entirely the enormous benefit of not needing crystals. I’m a (former) chemist that has only ever had to use X-ray crystallography for small molecules. I’m just trying to get a sense of what the practice of doing this is like?
- natechols 6y agoI do not know what it's like in practice now, but on a purely theoretical basis, if you are trying to solve a completely novel structure with no known homologs, especially if it's something on the scale of a ribosome, direct imaging is always going to be easier than trying to solve the phase problem, both experimentally and computationally.
- MayeulC 6y agoI 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.
- natechols 6y agoWith hard xrays you can visualize interatomic densities and lone pairs. I know there are structures down to 0.5Å resolution now but that must have required careful setup. I've seen diffraction out to 0.75Å but I could never collect complete data at that resolution.
- thechao 6y agoI’m imagining a collector plate the size of my dining room table, with a staff of crazy-haired scrawny physicists clambering around some sort of neo-Victorian rube-Goldbergesque X-ray source — like a wine-barrel-sized copper anode and a small nuclear power plant for the arc.
- natechols 6y agoThis is not far from the truth, except the detectors are actually relatively small (and very expensive), there's more aluminum foil than the Victorians used, and we have slightly better hair on average. The last time I used one of these facilities, it looked like this (and still does): https://lcls.slac.stanford.edu/sites/lcls.slac.stanford.edu/files/styles/basic_page_large_image/public/cxi-banner01.jpg https://lcls.slac.stanford.edu/sites/lcls.slac.stanford.edu/... Compared to this electron microscopy is downright boring - and a couple of orders of magnitude cheaper to set up.
- jtaillon 6y agoThat's pretty much correct. Single-atom imaging is possible using aberration-corrected scanning TEM, but the beam doses used will typically obliterate anything organic through radiolysis or ionization damage (although there are emerging ways around that using some interesting compressive sensing techniques). Most "atomic resolution" EM images you see are actually images of columns of atoms, since the imaging mode is transmission, so necessarily the image is summed through the plane of the sample (so atomic resolution is only in the perpendicular plane). The techniques used in this paper get around the dose issue by taking many images of very many presumably identical proteins (called dose fractionation) at very low dose. Computer algorithms are then used to stitch together a 3D model based on the low-dose individual images. Making it really cold also helps things from breaking down under the beam.
- __MatrixMan__ 6y agoPedantic 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.)
- dekhn 6y agoat 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 6y agoIndeed. 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 https://en.wikipedia.org/wiki/Atomic_nucleus for concrete example sizes.
- skosuri 6y agoPeople have used electron diffraction using cryo-em scopes to do sub-angstrom resolution. For example, my colleague Jose Rodridguez published this a few years ago. https://www.nature.com/articles/s41594-017-0018-0 https://www.nature.com/articles/s41594-017-0018-0
- dekhn 6y agoSee also neutron diffraction- it's been used for decades to get fine structure info.
- mensetmanusman 6y agoIt’s 7 orders for the protium isotope of hydrogen: https://www.wolframalpha.com/input/?i=diameter+of+hydrogen+atom+%2F+diameter+of+proton https://www.wolframalpha.com/input/?i=diameter+of+hydrogen+a... One reason hydrogen is so hard to see...
- hovden 6y agoThe 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.
- edna314 6y agoNaive 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?
- natechols 6y agoThe 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 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.)
- edna314 6y agoHmm, but then the b-factor (and therefore also resolution) is dependent on the flexibility of molecule you want to look at? Does that mean that these guys just found a system for which the main chain was rigid enough such that the thermal fluctuations are low enough?
- natechols 6y agoOh absolutely, they picked a model system that is both very rigid and has extremely high internal symmetry, and I assume they did rotational averaging to use fewer images. But these ideal systems are very useful for determining the limitations of the underlying technology like electron detectors; 15 years ago the best you could do with a molecule like this was around 4 Angstrom resolution.
- edna314 6y agoOk, that’s fair. But, then the number makes more sense in relative terms than in absolute. Still, absolutely impressive.
- deleted 6y ago[deleted]
- _Microft 6y agoSince 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 https://en.wikipedia.org/wiki/Angstrom
- vikramkr 6y agoCryo-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 https://www.nature.com/articles/nmeth.4115
- dekhn 6y agoIt'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.
- natechols 6y agoI 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.
- entee 6y agoSharing the OP’s position, and having worked in a biochemical technology development lab in grad school, I don’t think that’s quite right. Structural biology is disproportionately seductive as a data source because we are such visual creatures. You “see” it so it must be true. I’ve definitely been guilty of it while looking at protein structures, I doubt anyone in the field hasn’t. Hell there used to be 3D rigs you could buy with IR activated LED shutter glasses so you could see structures in 3D on your monitor and “better understand” the structure. It’s easy to get misled, proteins are incredibly dynamic in solution. Binding pockets appear and disappear, change shape, add and remove water. Given a structure of an unknown enzyme, with little to no biochemical characterization, you can’t tell what the catalytic mechanism or rate is. You probably can’t tell what the crucial residues are for binding at all. You may be able to tell the binding pocket, and... generate some hypotheses! That’s not to say protein structures aren’t helpful, they are! Especially things like co-crystals with drugs can help study that specific interaction. But I think the OP is totally right, we should back off strong conclusions on structure alone. It’s a great hypothesis generator, not a final answer in and of itself. The awesome part of cryoem at atomic scale is that we can now get MORE hypotheses generated faster with tougher proteins than with X-ray crystallography and other methods.
- Santosh83 6y agoSo 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 6y agoDo you mean the uncertainty principle or the observer effect? From Wikipedia: https://en.wikipedia.org/wiki/Uncertainty_principle 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) 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.
- tgbugs 6y agoUP 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 6y agoSo 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.
- alextheparrot 6y agoInteresting 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.
- natechols 6y agoYou 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.
- deleted 6y ago[deleted]
- fabian2k 6y agoThe 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 6y agoNext, 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 6y agoWe 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 https://youtube.com/watch?v=OuAGeQYjfus
- natechols 6y agoMost 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 6y agoWhat does the color information represent in the image?
- flobosg 6y agoSuch 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.
- natechols 6y agoIt's also possible it's colored by uncertainty in the atomic positions - what crystallographers call the "B-factor".
- flobosg 6y agoThat is true, but B-factors are mostly shown in a ribbon, cartoon, or "putty" (e.g. https://www.researchgate.net/figure/B-factor-diagram-of-protomeric-OmpU-represented-by-the-B-factor-putty-program-in-PyMOL_fig2_322190994 https://www.researchgate.net/figure/B-factor-diagram-of-prot...) representation, not as ball-and-sticks.
- aendruk 6y agoLooks like it's just distance from the center, probably for the sake of visualizing 3D space in a press release.
- flobosg 6y agoThat's another plausible option that I forgot to consider.
- sradman 6y agoThe 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 https://www.nature.com/articles/s41586-020-2833-4
- flobosg 6y agoThere 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 https://www.nature.com/articles/s41586-020-2829-0
- irjustin 6y agoCan 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.
- tgbugs 6y agoSome 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/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2359769/ 1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210420/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210420/
- DoofusOfDeath 6y agoLayman'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 6y agoNo, not by a long shot unfortunately.
- hobofan 6y agoEspecially 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).
- DoofusOfDeath 6y agoSo 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?
- hobofan 6y agoThe covalent bonds stay the same (which is why it's still the same molecule), apart for sometimes localized modification (e.g. protonation). The 3D structure (= shape) however, is mostly dependent on the hydrogen bonds (among other things like bisulfide bonds) between the amino acids that make up a protein. So the deprotonated variant of a amino acid side chain might not be able to interact in the same way as its protonated form, which will cause a different 3D structure for the whole protein, which in turn influences how it interacts with other molecules.
- 6y ago
- georgewfraser 6y agoWhen 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 6y agoWondering 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 6y agoThis 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.
- natechols 6y agoAcademic 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 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.)
- mmmBacon 6y agoSort 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 6y agoThe original paper: https://www.nature.com/articles/s41586-020-2829-0 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)