Solving protein nanocrystals by cryo-EM diffraction: Multiple scattering artifacts

Subramanian, Ganesh; Basu, Shibom; Liu, Haiguang; Zuo, Jian‐Min; Spence, John C. H.

doi:10.1016/j.ultramic.2014.08.013

Cited by 52 publications

(57 citation statements)

References 30 publications

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“…According to dynamical scattering theory (10), multiple scattering events produce inaccuracies in the recorded reflections, potentially preventing the solution of structures. Simulations suggest that with crystals thicker than 50-100 nm (11,12) dynamical scattering can be severe, resulting in nearly random intensities, where the relationship between the intensity and structure factor no longer holds true (10). However, these simulations assume diffraction is recorded from a perfect and stationary crystal -real macromolecular crystals are not perfect.…”

Section: Introductionmentioning

confidence: 97%

MicroED Structures from Micrometer Thick Protein Crystals

Martynowycz¹,

Glynn

Miao

et al. 2017

Preprint

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SummaryAtomic resolution protein structures can be determined by MicroED from crystals that surpass the theoretical maximum thickness limit by an order of magnitude. AbstractTheoretical 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.peer-reviewed)

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Section: Introductionmentioning

confidence: 97%

MicroED Structures from Micrometer Thick Protein Crystals

Martynowycz¹,

Glynn

Miao

et al. 2017

Preprint

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“…Because MicroED is capable of producing atomic resolution data, direct methods approaches used in X-ray crystallography should be applicable to MicroED. The influence of dynamical scattering on the measured intensities was thought to limit accurate determination of structure factors (23)(24)(25)(26). The hypothesis of whether these effects limit ab initio determination of macromolecular structures from protein nanocrystals has been difficult to test because a crystal formed from macromolecules has yet to diffract to better than 1.2 Å by electron-based techniques.…”

mentioning

confidence: 99%

Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED

Sawaya

Rodríguez

Cascio

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

104

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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.

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“…Dynamical scattering can compromise structure solution of crystals of macromolecules, since current phasing methods and refinement procedures do not account for its effects. Thin crystals minimize the effects of dynamic scattering, and on the basis of multi-slice simulations it has been suggested that the maximal thickness of a protein crystal that still allows structure solution is about 100 nm for 200 keV electrons (Subramanian et al, 2015), but these calculations ignore inelastic scattering, which is three times more prevalent than elastic diffraction for organic samples.…”

Section: Discussionmentioning

confidence: 99%

Protein structure determination by electron diffraction using a single three-dimensional nanocrystal

Clabbers

Genderen

Wan

et al. 2017

Acta Crystallogr D Struct Biol

100

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Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of only 0.14 mm 3 , i.e. no more than 6 Â 10 5 unit cells, provided sufficient information to determine the structure of a rare dimeric polymorph of hen egg-white lysozyme by electron crystallography. This is at least an order of magnitude smaller than was previously possible. The molecular-replacement solution, based on a monomeric polyalanine model, provided sufficient phasing power to show side-chain density, and automated model building was used to reconstruct the side chains. Diffraction data were acquired using the rotation method with parallel beam diffraction on a Titan Krios transmission electron microscope equipped with a novel in-house-designed 1024 Â 1024 pixel Timepix hybrid pixel detector for low-dose diffraction data collection. Favourable detector characteristics include the ability to accurately discriminate single high-energy electrons from X-rays and count them, fast readout to finely sample reciprocal space and a high dynamic range. This work, together with other recent milestones, suggests that electron crystallography can provide an attractive alternative in determining biological structures.

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Solving protein nanocrystals by cryo-EM diffraction: Multiple scattering artifacts

Cited by 52 publications

References 30 publications

MicroED Structures from Micrometer Thick Protein Crystals

MicroED Structures from Micrometer Thick Protein Crystals

Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED

Protein structure determination by electron diffraction using a single three-dimensional nanocrystal

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