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 as many membrane proteins. Careful crystal pre-characterization including compatibility testing of the sample delivery method is essential to ensure efficient use of the limited beamtime available at XFEL sources. This work demonstrates the utility of transmission electron microscopy 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 transform of 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) the multi-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.

Goniometer-based femtosecond crystallography with X-ray free electron lasers

“…Diffraction patterns of superior resolution (3.3 Å) were observed when using the microdiffractometer setup at LCLS XPP at 100 K than were previously measured at LCLS-CXI or at the synchrotron (Table S2). The lower diffraction resolution observed (4.0 Å) during screening experiments at LCLS CXI using smaller crystals and the GDVN injector suggests that a larger sample volume may be required to obtain higher resolution diffraction, or that the crystals were damaged during injection, potentially a consequence of their high solvent content (24). The best diffraction resolution (3.7 Å) observed at SSRL BL12-2 from these crystals (grown in same crystallization batch as used at LCLS XPP), suggests that Pol II-TFIIB-NAS crystals suffer significant radiation damage during the exposure time needed to deliver an equivalent dose at a synchrotron.…”

“…However, there are different and often more complex optimization steps associated with specific injector technologies. Solutions containing a mixture of crystal sizes may require filtering to avoid clogging in the injector nozzle, and delicate crystals may be damaged from the pressures and shear forces of the delivery process itself (24). For experiments conducted in vacuo, stream formation may be disrupted by solution bubbling, drying, or freezing as it exits the injector and enters the vacuum chamber.…”

The birth of a new field

“…It was immediately realized that the high spatial coherence provided new approaches to the phase problem, for both SPs (Loh [7], Ourmazd and co-workers [8], Martin [9], Schwander et al [16]) and nanocrystals (Millane & Chen [17], Kirian et al [18], Spence et al [19] and Barty et al [20]). Atomic resolution has so far been obtained from unknown structures only using nanocrystals (and near-atomic resolution in the FSS when small changes in a known structure are studied), so that a crucial area for development of the BioXFEL field is the development of new methods for growing nanocrystals (Kupitz et al [21], Caffrey et al [22], Gallat et al [23] and Stevenson et al [24]). When the study of the evolving damage processes (reviewed by Chapman et al [3]), diffraction physics (White [25]), simultaneous emission spectroscopy (Kern et al [15]) and detector development (Denes [26]) is added to this list of subfields, it will be seen that structure and dynamics in biology with XFELs is an extremely rich interdisciplinary field, now undergoing rapid innovation and creative development.…”

Serial femtosecond crystallography opens new avenues for Structural Biology

“…The most important characteristics in this optimization are size distribution, density and, of course, diffraction quality. Size distribution for submicron crystals can be obtained using dynamic light scattering [29,45], Nanosight [33,46] or electron microscopy [47]. A new microfluidic technique even allows inert post crystallization sorting using dielectrophoresis [35].…”

“…A new microfluidic technique even allows inert post crystallization sorting using dielectrophoresis [35]. Electron microscopy is an excellent characterization tool for nanocrystals, allowing predicted diffraction quality, analysis of size distribution and permitting comparison of samples at various stages in the sample preparation and subsequent experiment [47] (see Fig. 4).…”