Fixed-target protein serial microcrystallography with an x-ray free electron laser

Abstract

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Protein Micro-Crystallography: Nanotechnology Challenges Ahead

“…Indeed, an LCP matrix filled with about 30 μm size bacteriorhodopsin crystallites was extruded into air and probed by SSX [35].This membrane protein structure was refined to 0.24 nm resolution. A similar protein consumption has been obtained for randomly distributed crystallites on a fixed target by raster-scan SFX [32]. The same approach was used for SSX with micro-and nanobeams on a slurry of lysozyme crystallites contained between Si 3 N 4 membranes ( Figure 5A & 5B).…”

“…A number of room temperature sample environments tested at SR-MPX beam lines have been initially developed for SFX experiments [12,[30][31][32][33]. Such experiments are generally performed without crystal rotation.…”

DA+ data acquisition and analysis software at the Swiss Light Source macromolecular crystallography beamlines

“…Users can configure the ROI in the DA+ GUI for both standard data collection (default is 16 M) and grid scan (default is 4 M). The 4 M ROI is particularly suitable for fast grid scans on large samples with multiple microcrystals, such as solid supports (Hunter et al, 2014;Feld et al, 2015) and in meso in situ serial crystallography (IMISX) plates (Huang et al, 2015 as the 4 M images are evaluated in a fraction of the time needed for a 16 M image. An example of a grid scan performed on a microcrystal with the microbeam at 50 Hz is shown in Fig.…”

A Graphene-Based Microfluidic Platform for Electrocrystallization and In Situ X-ray Diffraction

“…Such platforms have been increasingly harnessed to facilitate the diffraction analysis of challenging targets for both static and dynamic structure determination. Various platforms have been developed to improve the growth and subsequent mounting of tiny and fragile crystals for X-ray diffraction analysis [24][25][26][27][28], including dense array-style devices [29][30][31][32][33][34][35][36][37][38][39][40][41], platforms for the lipidic cubic phase crystallization of membrane proteins [42,43], and thin-film sandwich devices [44]. In the meantime, the challenges of such platforms lie in the need to maintain a protected sample environment, as well as minimize the interference of device materials with the subsequent X-ray analysis.…”