Transmission electron microscopy as a tool for nanocrystal characterization pre- and post-injector

Abstract

Recent advancements at the Linac Coherent Light Source X-ray free-electron laser (XFEL) enabling successful serial femtosecond diffraction experiments using nanometre-sized crystals (NCs) have opened up the possibility of X-ray structure determination of proteins that produce only submicrometre crystals such asmanymembrane proteins. Careful crystal pre-characterization including compatibility testingof the sample deliverymethodis essential to ensure efficient use of the limited beamtime available at XFEL sources. This work demonstrates the utility of transmission electronmicroscopy for detecting and evaluating NCs within the carrier solutions of liquid injectors. The diffraction quality of these crystals may be assessed by examining the crystal lattice and by calculating the fast Fourier transformof the image. Injector reservoir solutions, as well as solutions collected post-injection,were evaluated for three types of protein NCs (i) the membrane protein PTHR1, (ii) themulti-protein complex Pol II-GFP and (iii) the soluble protein lysozyme.Our results indicate that the concentration and diffraction quality of NCs, particularly those with high solvent content and sensitivity to mechanical manipulation may be affected by the delivery process. © 2014 The Author(s) Published by the Royal Society. All rights reserved.