@article {48265, title = {Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.}, journal = {Nature Methods}, volume = {14}, year = {2017}, month = {2017 Feb 13}, pages = {399-402}, abstract = {

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.

}, issn = {1548-7105}, doi = {10.1038/nmeth.4178}, author = {de la Cruz, M Jason and Hattne, Johan and Shi, Dan and Seidler, Paul and Rodriguez, Jose and Reyes, Francis E and Sawaya, Michael R and Cascio, Duilio and Weiss, Simon C and Kim, Sun Kyung and Hinck, Cynthia S and Hinck, Andrew P and Calero, Guillermo and Eisenberg, David and Gonen, Tamir} } @article {48603, title = {MicroED structures from micrometer thick protein crystals.}, journal = {bioRxiv}, year = {2017}, month = {2017 Jun 20}, abstract = {

Theoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals. Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments.

}, doi = {10.1101/152504}, author = {Martynowycz, Michael and Glynn, Calina and Miao, Jennifer and de la Cruz, Michael Jason and Hattne, Johan and Shi, Dan and Cascio, Duilio and Rodriguez, Jose and Gonen, Tamir} } @article {47875, title = {Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {113}, year = {2016}, month = {2016 Oct 04}, pages = {11232-6}, abstract = {

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.

}, issn = {1091-6490}, doi = {10.1073/pnas.1606287113}, author = {Sawaya, Michael R and Rodriguez, Jose and Cascio, Duilio and Collazo, Michael J and Shi, Dan and Reyes, Francis E and Hattne, Johan and Gonen, Tamir and Eisenberg, David S} }