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Analytical Chemistry

Down to the Atoms

I wanted to mention something that was reported a week or so ago, and may sound a bit exotic or obscure, if you’re not a structural biologist. But it’s yet another sign of a revolution in our ability to get structures of biomolecules (and others) that we never would have before, and the effects over the coming years are going to be profound.

I’m talking about the improvement in cryo-electron microscopy to get down to atomic resolution. I’ve spoken about cryo-EM several times before on the blog (most recently here and also elsewhere), since over the lifetime of the blog itself the technique has made huge advances. Now two papers in Nature report the latest, which takes the quality of cryo-EM data to a level that twenty years ago not many people would have ever expected to see.

The early 2010s brought a “resolution revolution” that isn’t over yet. It’s been a combination of several techniques and improvements in both hardware and software. Cryo-EM can be computationally intensive, so even if the hardware had been available in (say) 1985, we wouldn’t have been able to extract the information out of it that we can now (at least not in any useful amount of time!) But it’s not all just better processors and better software; the newer hardware is no small part of the story. We have better electron sources, better ways to keep the energies of the beam within a very narrow range and very precisely aimed, better detectors at the back end of the sample, new noise-reduction techniques in general – all of these and more have combined with all that software power to produce something amazing.

If you’re wondering what the big deal is, it might be summed up as “you don’t always have to grow crystals any more”. X-ray crystallography has been a huge technique ever since its beginnings early in the 20th century, and has advanced over the decades for some of the same reasons that cryo-EM has more recently: better X-ray sources (both brighter and tighter), better detectors, and better algorithms and hardware to run them on. Modern crystallography would floor the folks who were doing huge amounts of work to generate every single structure back in the 1950s. But you’ve still needed crystals, high-quality ones, to get the technique to work. And growing them is witchcraft. Really, it is. As I’ve said many times, all you have to do is walk into a protein crystallography lab and see the stacks of multiwell plates everywhere, full of dozens, hundreds, thousands of attempts to grow good crystals by varying the concentrations, varying the buffers, varying the protein constructs, varying the long, long list of salts and counterions and additives that people have tried over the years to coax the proteins into somehow lining up and forming orderly ranks.

As anyone who’s done the slightest bit of work in the field will attest, even once you see beautiful chunky crystals forming – and not those fluffy threads, those usually don’t work out – you’re nowhere near out of the woods. The first big question is “Is that actually your protein?” because those salts and additives can form nice crystals of their own, too, and the second big question is “Does it diffract?” Very decorative-looking crystals can turn out to be crap when the X-ray beam hits them: what you want is showers of well-defined diffraction spots, varying in sweeps of beautiful patterns as the orientation changes between the crystal and the beam, and stretching way out to the edges of your detector (the further out they go, the higher the resolution you will likely be able to wring out of the eventual data set). What you get, too often, is splorts and splotches, smeary gorp that starts out ugly and gets uglier as you watch. Robert Palmer warned us years ago that a pretty face don’t mean no pretty heart, and every crystallographer knows that he was right. Why yes, I have tried growing crystals and collecting data on them personally: did I give myself away?

But what if you didn’t have to do this? Cryo-EM doesn’t need crystals. You take your protein of interest and spread a solution of it out on a surface, which after a few more steps gives you a scattering of individual protein particles lying there every which way. And you hit them individually with your fancy electron beam and collect the results on your fancy detector, with all that data going into some very fancy software indeed. That software can, if all goes well, sort the particles you collected on into categories – edgewise, tilted, right down the middle, lying on the long side, and so on – and build a model of what the protein must look like that could have generated the data. Used to be, this process gave you fuzzballs, but they’ve been getting relentlessly sharper.

And now we’re down to individual atoms, which is what a high-quality X-ray structure has always been able to deliver. As the resolution of these techniques improves, the pictures become startling. An aromatic ring, for example, like a phenylalanine side chain on a protein, is a fuzzy blob with lousy data. Better data show you that it’s a flattened blob, and you can start to see how it’s tilted in three dimensions. Produce higher-quality stuff, and that aromatic ring stops looking like a hexagonal lump of wood and starts becoming a hexagonal doughnut: you’re seeing the open space in the center of the six-membered ring. That level is very nice resolution for a protein structure, but the small-molecule crystallographers can push on to even more. Higher resolution still, and that doughnut turns into a ring made from six ball bearings: the individual carbon atoms. Beyond that, you can even start to see ghostly electron density between them, which is the shared electrons of the chemical bonds themselves. You can see the improvement in the real-world structures below, from this review article. The ball-and-stick stuff is the model, and the meshwork is what you actually get:

Note that the highest-resolution structures there are rare and difficult. But these two new cryo-EM papers have a well-behaved protein (apo-ferritin) resolved to just over 1.2 Ångstrom, and a membrane protein (a GABA receptor) at 1.7Å. That’s really damned good, and the exciting thing is that the technology is still improving. This overview at Nature suggests that we may be getting near the end improving the electron beams and the like, but that there’s plenty of room in sample preparation and data analysis. This will allow us to see more and more detail on progressively harder and harder to handle samples.

Consider cryo-electron tomography, where you apply these techniques to proteins in the cells themselves. This is a pretty intense technique, but it’s already providing insights into protein structure and function that we couldn’t get any other way. This stuff would have been considered impossible not all that long ago, and I’m very happy to be able to see it happening for real.

22 comments on “Down to the Atoms”

  1. Simon AuclairtheGreatandTerrible says:

    Neat! Maybe we’ll get x ray 3d holography next.

  2. Athaic says:

    “all you have to do is walk into a protein crystallography lab and see the stacks of multiwell plates everywhere”

    Uh. In a project I was involved with, the crystallographer colleagues announced in the penultimate meeting before the end of the project that they had finally managed to acquire a X-ray of the crystal of our protein.
    As they told us, they “just picked-up and looked again at a vial with the protein in solution that they have left sitting on a shelf for about six months”.
    At the time, I found them a bit flippant about it.
    Now I’m starting to understand the unsaid parts. There likely was plenty more vials “left sitting on a shelf” involved in the story. More shelves, too.

  3. Hugo says:

    I continue to be impressed by the resolution, especially considering the amount of wet work involved (I have no idea about the computational part). Can anyone here give their opinion about progress towards structures of smaller proteins? These are some huge complexes, 500 and 250 kDa. Any chance we’ll get this working for 50 kDa proteins?

    1. ryan shaw says:

      Hugo, we are already there, with ~3A structures of GPCRs.

      There are a huge number of unsolved problems, and sample preparation challenges, but open questions are as always, enticing and exciting.

      The next step is to resolve these structures in cellular systems, to see how function is occurring in vivo.

      We will need some continued development in the space of sample preparation and freezing, but proof of concept is there already.

  4. Barry says:

    Since Max Perutz, we’ve wondered whether the conformation we see in x-ray diffraction is a major conformation in solution, or merely one selected by crystal packing forces. Now, with cryo-EM, we can instead wonder whether the conformation we’re seeing is on favored by contact with the (copper?) surface.
    But we now have piles of NMR data showing that the x-ray structures usually do correspond to solution structure

    1. ccm says:

      The protein of interest isn’t imaged close to the copper in the grid. Instead, it’s usually embedded in vitreous ice within a small carbon hole. However, particles are often exposed to atmosphere at the edge of the ice, which is definitely also not physiological. There’s a video showing this here: https://vimeo.com/369583017

      1. Barry says:

        Many thanks! I had misunderstood the open grid.

  5. Konstantin Korotkov says:

    Streptavidin – 52kDa – is probably the smallest protein structure determined by single-particle cryoEM to high resolution: https://www.nature.com/articles/s41467-019-10368-w

    1. Grouchy says:

      Importantly, 3.2 A is not the same as 1.7 A.

  6. Chris Phoenix says:

    What about LEAP microscopy?
    https://www.nist.gov/laboratories/tools-instruments/local-electrode-atom-probe-tomography-leap
    has a picture of 80x80x180 nm volume and they can get sub-nm accuracy and tell you which atom type came from where. It works on conductors, semiconductors, and insulators.

    Seems like that might be extended to view proteins in an amorphous dried clump? I’m assuming cryo-EM is better, but it’s not the only way to image individual atoms in 3D.

    1. Derek Lowe says:

      Ah, that one’s a different beast. It’s blasting things off to analyze by mass spec, and can handle impressively small volumes with high accuracy. But if you tried that on a protein sample, you would just destroy the structure along the way, and get a readout, across the whole sample, of mostly carbon atoms with some nitrogen, oxygen, and the occasional sulfur atom flung out into the mass spec detector. But the 3D structure would be lost.

  7. The thing that perhaps gets me most excited about this is actually how complementary cryo-EM and x-ray crystallography are. Cryo-EM tends to be strongest exactly where crystallography is weak and vice versa. Cryo-EM does best on great big complexes but really struggles to find and align smaller ones; crystallography is generally most successful on small, fairly rigid proteins. On big complexes cryo-EM tends to get the rigid core really clear, but falls apart on the periphery where you often have flexible or weakly-attached domains flopping around – often exactly the sort of things you could chop out and solve successfully using crystallography to construct a best-of-both-worlds hybrid model.

    Where crystallography really still holds its own, though (and probably will for quite some time yet) is fragment screening. Typically, once you have *a* crystal you can have as many crystals as you can afford to produce the protein for, making high-throughput soaking experiments both cheap and fast. It’s hard to see an equivalent in cryo-EM (although I’m open to surprises)!

  8. Ogamol says:

    So, in the pictured example .55Å shows the atoms and 1.5 Å gives us electron cloud densities. (The points of paucity suggests questions of connectivity.)

  9. 10 Fingers says:

    My favorite term, from those I have worked with who are true practitioners-of-the-art in every sense, when in the middle of the stochastic journey that is protein crystallography:

    “Promising precipitate”

    I’m looking forward to finding out some of the cryo-EM equivalents.

  10. geppy says:

    @Derek , you always had a weakness for Traditional Chinese Medicine. You should do a review of this amazing article, just published by Jean Nachega at University of Pittsburgh:

    Can Traditional Chinese Medicine provide insights into controlling the COVID-19 pandemic: Serpentinization-induced lithospheric long-wavelength magnetic anomalies in Proterozoic bedrocks in a weakened geomagnetic field mediate the aberrant transformation of biogenic molecules in COVID-19 via magnetic catalysis

    https://www.sciencedirect.com/science/article/pii/S0048969720363592#s0100

    1. Derek Lowe says:

      Good God, you must be kidding. Elsevier has way too many journals, and publishes way too much crap.

    2. Marko says:

      ” This work was funded through grants from the United States-National Institutes of Health ”

      I’m all for govenrnent-funded basic research , but this might be a bit of a stretch.

      1. zero says:

        Thank senator Hatch among others for supporting stuff like that.

        I think we should be willing to test just about anything as long as we can do it rigorously and will publish the results either way. I also think we should stop funding things (like homeopathy) that are proven not to work.

    3. Nick says:

      Wow,
      That is a toss up between an elaborate April Fools joke and propaganda.

    4. fajensen says:

      Whenever a headline is stated as a question, the answer is “No”!

  11. David Edwards says:

    It’s articles like this that keep me coming back to read more, while waiting for the TIWWW entries to expand.

    Derek, you have more of a talent for making complex science such as this comprehensible, than you probably give yourself credit for.

    Now I’m enjoying the hilarity of wading through the papers themselves … which, thanks to this article, will be somewhat less intimidating …

  12. Roger Callaway says:

    My Mom worked as a lab tech in the 30’s and 40’s stain cells. She spoke of staining the”Red-Eyed Bugs of Love”, Gonorrhea bacteria. She would be amazed.

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