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

Abstract

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.

Solving the first novel protein structure by 3D micro-crystal electron diffraction

“…Due to the strong interactions between electrons and matter, crystals that are considered as powder in X-ray crystallography can be treated as single crystals by micro-crystal electron diffraction (MicroED (Shi et al, 2013)). This enables structure determination of molecules from micron-to nanometer-sized 3D crystals that are too small for conventional X-ray diffraction (Shi et al, 2013;Nannenga, Shi, Leslie et al, 2014;Yonekura et al, 2015;Clabbers et al, 2017;Xu et al, 2018). Furthermore, MicroED can be applied to study biomolecules of low molecular weight that are beyond what can be resolved by single particle cryo-EM imaging (Henderson, 1995;Khoshouei et al, 2017).…”

Monitoring the production of high diffraction-quality crystals of two enzymes in real time usingin situdynamic light scattering

“…The recent advent of X-ray Free electron lasers (X-FEL) enables the serial femtosecond diffraction (SFX) analysis of micro-and nano-crystals, and offers unprecedented possibilities for time-resolved experiments [2][3][4]. At the same time, electron microscopes have been highjacked to perform micro-electron diffraction (µED), opening the way to the characterization of nanocrystals using laboratory-based instruments [5][6][7][8]. Though, like conventional ones relying on synchrotron or neutron sources, these new crystallographic approaches require crystalline material and call for the development of means facilitating the production of calibrated samples (i.e.…”

Serial protein crystallography in an electron microscope

“…While seminal experiments on 2D crystals 20 were restricted to a small class of suitable samples, various successful implementations of 3D rotation electron diffraction (3D ED) solving structures of beam-sensitive small molecules [21][22][23] sparked interest in applying 3D crystallography also to biomolecules, a technique also referred to as MicroED [24][25][26] . Several research groups have now succeeded in solving protein structures by merging electron diffraction data from as little as one up to a few sub-micron sized vitrified protein crystals using rotation diffraction [27][28][29][30] , and very recently the first unknown protein structure could be determined 31 . Automated procedures are becoming increasingly available to reduce the manual effort of identifying suitable crystals, acquiring rotation series while keeping the crystal under the beam, and sequentially addressing many crystals to be merged [32][33][34][35][36] .…”