Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals.

Alexander M Wolff,Tamir Gonen,Michihiro Sugahara,Kazutaka Ito,Avi J Samelson,Fumiaki Yumoto,Kensuke Tono,Asmit Bhowmick,Andrew Aquila,Michael W Martynowycz,Takashi Nomura,Shigeki Owada,Dohyun Im,Raymond G Sierra,Sergio Carbajo,Michael C Thompson,So Iwata,Takanori Nakane,Aaron S Brewster,Minoru Kubo,Nicholas K Sauter,Aina E Cohen,Tomas S Lazarou,Wei Zhao,James S Fraser,Eriko Nango,Tomoyuki Tanaka,Tsuneo Hino ,Jake Koralek ,I.d Young ,R.a Woldeyes ,J.t Biel ,Erin Thompson ,H Van Den Bedem ,R Tanaka ,S.v Cortez ,Sébastien Boutet ,Ana González

doi:10.1107/s205225252000072x

Abstract

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

Highlights

2015); these crystals are too large for precipitant led to increased crystal density while maintaining either microfluidic serial crystallography or microcrystal electron diffraction (MicroED)
As a first step towards this goal, final PEG 3350 concentration was near 20% (Fig. 1). we systematically explored the phase space of cyclophilin A (CypA) crystal- Increasing the protein concentration beyond 35 mg mlÀ1 did lization in the vicinity of the conditions that yield large crystals not lead to appreciable increases in crystal density, so we [protein concentration in the range 80–100 mg mlÀ1 with 20– chose a final protein concentration of 35 mg mlÀ1 and a final
The work that we present here attempts to address this knowledge gap by providing a detailed description of how we optimized the growth of cyclophilin A (CypA) microcrystals and measured their diffraction using two emerging microcrystallography techniques: serial X-ray free-electron lasers (XFELs) crystallography and microcrystal electron diffraction (MicroED)

Summary

Introduction

Small crystals are advantageous when the diffraction experiment is preceded by a perturbation to the crystal This includes common crystal treatments such as flash-cooling or ligand soaking, as well as more uncommon perturbations such as the stimulation of crystallized molecules for timeresolved experiments (Coquelle et al, 2018; Olmos et al, 2018). Because they have substantially less volume and a limited number of unit cells, perturbations can be applied more rapidly and uniformly to smaller crystals than to larger crystals, and smaller crystals accumulate less strain resulting from changes in crystal lattice dimensions. The development of protein ‘microcrystallography’ techniques, which are optimized for measuring crystals with physical dimensions of tens of micrometres or smaller, has offered access to these opportunities and benefits, and has led to a shift in what is considered to be a valuable specimen for experimental characterization

Methods

Results

Conclusion