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Analytical Chemistry

A New Way to Get Protein Crystal Structures

There’s a report of a new technique to solve protein crystal structures on a much smaller scale than anyone’s done before. Here’s the paper: the team at the Howard Hughes Medical Institute has used cryo-electron microscopy to do electron diffraction on microcrystals of lysozyme protein.

We present a method, ‘MicroED’, for structure determination by electron crystallography. It should be widely applicable to both soluble and membrane proteins as long as small, well-ordered crystals can be obtained. We have shown that diffraction data at atomic resolution can be collected and a structure determined from crystals that are up to 6 orders of magnitude smaller in volume than those typically used for X-ray crystallography.
For difficult targets such as membrane proteins and multi-protein complexes, screening often produces microcrystals that require a great deal of optimization before reaching the size required for X-ray crystallography. Sometimes such size optimization becomes an impassable barrier. Electron diffraction of microcrystals as described here offers an alternative, allowing this roadblock to be bypassed and data to be collected directly from the initial crystallization hits.

X-ray diffraction is, of course, the usual way to determine crystal structures. Electrons can do the same thing for you, but practically speaking, that’s been hard to realize in a general sense. Protein crystals don’t stand up very well to electron beams, particularly if you crank up the intensity in order to see lots of diffraction spots. Electrons interact strongly with atoms, which is nice, because you don’t need as big a sample to get diffraction, but they interact so strongly that things start falling apart pretty quickly. You can collect more data by zapping more crystals, but the problem is that you don’t know how these things are oriented relative to each other. That leaves you with a pile of jigsaw-puzzle diffraction data and no easy way to fit it together. So the most common application for protein electron crystallography has been for samples that crystallize in a thin film or monolayer – that way, you can continue collecting diffraction data while being a bit more sure that everything is facing in the same direction.
lysozyme crystals
In this new technique, the intensity of the electron beam is turned down greatly, and the crystal itself is precisely rotated through 90 one-degree increments. The team has developed methods to handle the data and combine it into a useful set, and were able to get a 2.9-angstrom resolution on lysozyme crystals that are (as described above) far smaller than the usual standard for X-ray work, as shown. There’s been a lot of work over the years to figure out how low you can set the electron intensity and still get useful data in such experiments, and this work started off by figuring out how much total radiation the crystals could stand and dividing that out into portions.
The paper, commendably, has a long section detailing how they tried to check for bias in their structure models, and the data seem pretty solid, for what that’s worth coming from a non-crystallographer like me. This is still a work in progress, though – lysozyme is about the easiest example possible, for one thing. The authors describe some of the improvements in data collection and handling that would help make this a regular structural biology tool, and I hope that it does so. There’s a lot of promise here – being able to pull structures out of tiny “useless” protein crystals would be a real advance.

2 comments on “A New Way to Get Protein Crystal Structures”

  1. David Borhani says:

    The decision letter (online at end of article) is very informative vis-a-vis the promise and limitations of this method. Worth reading.

  2. Ivan Bushmarinov says:

    While not a protein crystallographer myself, I must note that the choice of the journal says very much about the quality of the work here. Such data should have been published in either Acta Cryst D, J. Applied Crystallography or the fresh and hip IUCrJ — if the results could stand up to the scrutiny of the professional community.
    The other point worth noting is that X-ray diffraction on nanocrystals using free-electron lasers (XFEL) is the modern, widely discussed and in many ways superior approach to the problem. These guys are just late.

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