Nanoscale mosaicity revealed in peptide microcrystals by scanning electron nanodiffraction

Marcus Gallagher-Jones,Ouliana Panova,Andrew M Minor,Calina Glynn,Jose A Rodriguez,Jim Ciston,Colin Ophus,Kevin Canton Mancia,David R Boyer,Chih-Te Zee,Karen C Bustillo

doi:10.1038/s42003-018-0263-8

Abstract

Changes in lattice structure across sub-regions of protein crystals are challenging to assess when relying on whole crystal measurements. Because of this difficulty, macromolecular structure determination from protein micro and nanocrystals requires assumptions of bulk crystallinity and domain block substructure. Here we map lattice structure across micron size areas of cryogenically preserved three−dimensional peptide crystals using a nano-focused electron beam. This approach produces diffraction from as few as 1500 molecules in a crystal, is sensitive to crystal thickness and three−dimensional lattice orientation. Real-space maps reconstructed from unsupervised classification of diffraction patterns across a crystal reveal regions of crystal order/disorder and three−dimensional lattice tilts on the sub-100nm scale. The nanoscale lattice reorientation observed in the micron-sized peptide crystal lattices studied here provides a direct view of their plasticity. Knowledge of these features facilitates an improved understanding of peptide assemblies that could aid in the determination of structures from nano- and microcrystals by single or serial crystal electron diffraction.

Highlights

Changes in lattice structure across sub-regions of protein crystals are challenging to assess when relying on whole crystal measurements
In contrast to semi-crystalline polymers studied previously by 4DSTEM41,42, the prion peptide nanocrystals we evaluated are composed of highly ordered peptide arrays that diffract to sub-ångstrom resolution[33]
Our measurement of 1.4 Å resolution diffraction from sub-10 nm regions of peptide crystals was facilitated by three key technological features of our experiment: fast readout direct electron counting detectors[62], a hybrid counting protocol applied to sparse diffraction data captured by 4DSTEM and low-dose cryogenic techniques that lessen the evidence of radiation damage

Summary

Introduction

Changes in lattice structure across sub-regions of protein crystals are challenging to assess when relying on whole crystal measurements. The nanoscale lattice reorientation observed in the micron-sized peptide crystal lattices studied here provides a direct view of their plasticity Knowledge of these features facilitates an improved understanding of peptide assemblies that could aid in the determination of structures from nano- and microcrystals by single or serial crystal electron diffraction. Because directly measuring mosaicity in protein crystals is inherently challenging[2], crystallographic software must estimate disparities in domain block size, shape and orientation per crystal[5,6,7], for full and partial Bragg reflections[5,8]. Domain blocks can be identified in cryo-EM images of three-dimensional (3D) lysozyme microcrystals[20], where Fourier filtering helps estimate the location and span of multiple blocks across a single crystal[20]

Methods

Results

Conclusion