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

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.

Atomic resolution structure determination by the cryo‐EM method MicroED

“…This method has yielded the structure of the water channel aquaporin‐0 and its surrounding membrane at 1.9 Å resolution from 2D crystals only a single protein layer thick . Finally, in MicroED vanishingly small three‐dimensional (3D) crystals of biological material are studied by electron diffraction under cryogenic conditions to reveal unprecedented atomic detail such as the positions of protons in a protein . In this review, we highlight the unique strengths of MicroED and summarize current advances in this method.…”

Analysis of Diffracted Intensities from Finite Protein Crystals with Incomplete Unit Cells

“…SXC experiments on finite protein crystals require the merging of diffraction data from large quantities of crystals to address experimental limitations imposed by radiation damage and for the collection of three-dimensional structural information. Diffraction data collected in microED experiments have also been reported to be merged from multiple crystals in some instances [24,25]. As derived by Kirian et al [13], the diffracted intensity distribution averaged from n finite crystals can be expressed as…”

Understanding pre-mRNA splicing through crystallography

“…Possible phasing methods include MIR method using heavy atoms, phasing through single particle cryo-EM maps, or direct phasing when resolution is high (close to 1Å). There are over a dozen structures determined by electron crystallography now 63 , four of which (prion peptides) were determined to 1 Å resolution using the direct phasing method 64 . This number is likely to increase exponentially as the technique continues to improve.…”