Serial femtosecond crystallography

Abstract

With the advent of X-ray Free Electron Lasers (XFELs), new, high-throughput serial crystallography techniques for macromolecular structure determination have emerged. Serial femtosecond crystallography (SFX) and related methods provide possibilities beyond canonical, single-crystal rotation crystallography by mitigating radiation damage and allowing time-resolved studies with unprecedented temporal resolution. This primer aims to assist structural biology groups with little or no experience in serial crystallography planning and carrying out a successful SFX experiment. It discusses the background of serial crystallography and its possibilities. Microcrystal growth and characterization methods are discussed, alongside techniques for sample delivery and data processing. Moreover, it gives practical tips for preparing an experiment, what to consider and do during a beamtime and how to conduct the final data analysis. Finally, the Primer looks at various applications of SFX, including structure determination of membrane proteins, investigation of radiation damage-prone systems and time-resolved studies.

Fig. 1 |. Differences in experimental crystallography setups.

a, c | conventional rotation crystallography. b, d | SFX with sample delivery by high viscosity extrusion. In conventional MX, a single crystal (red) is mounted in a loop, kept at 100 K using a cryogenic nitrogen stream and rotated during sequential exposures, panel a. Consecutively acquired diffraction patterns are indexed, giving an orientation matrix of the crystal in the laboratory coordinate system, which is used to integrate the reflection intensities, panel c. In SFX, many microcrystals are sequentially delivered to the pulsed X-ray beam in random orientations, and a detector image is acquired for each XFEL pulse, panel b. A Bragg diffraction pattern containing partial intensities will only be produced when a crystal is present in the interaction region at the same time as an X-ray pulse arrives, panel d. Images containing a diffraction pattern are selected in a process called “hit finding”. The hit rate is the ratio of pattern-containing frames to the total number of collected frames. Hits are individually indexed, which is not always successful. The ratio of indexed patterns to the total number of hits is the indexing rate, panel d. Individual indexed diffraction patterns are integrated, and the resulting intensities are merged by Monte-Carlo integration.

Fig. 2 |. SFX sample delivery methods.

a | Gas dynamic virtual nozzle (GDVN). A suspension of crystal-containing liquid is pumped through an inner capillary, surrounded by an outer capillary through which a gas flows (dashed arrows). At the end of the GDVN, the outer capillary constricts to narrow the gas stream, accelerating both gas- and liquid. The liquid stream is focused into a narrow jet that breaks up into droplets. b | High viscosity extrusion (HVE) injector. Crystals are dispersed in a highly viscous medium and slowly extruded into a stream. A gas sheath prevents the stream from curling back onto itself. c | Microfluidic Electrokinetic Sample Holder (MESH)^,. A high voltage (several kV) stretches a thin, slow-flowing stream of crystal-containing liquid between two electrodes. d | Serial Femtosecond Rotation crystallography (SF-ROX). A goniometer-mounted large crystal is translated and rotated between XFEL exposures. e | Drop-on-demand (DoD). Droplets of a crystal suspension are generated with, for example, a piezoelectric device. The droplet can be synchronized with and intersected by the XFEL pulse in free fall, immersed in an oil stream, or as shown in panel d, be deposited onto a tape and moved through the XFEL beam, Drop-on-Tape (DoT). f | Solid support methods. Crystals are deposited onto an X-ray transparent substrate, often referred to as a fixed target or chip, and scanned through the beam. Both unpatterned chips, on which crystals will assume random positions, and patterned chips (inset), which have wells for crystal location, are used.

Fig. 3 |. Distribution of sample delivery techniques used for published SFX experiments resulting in PDB codes.

Data up to December 2021. Of a total of 417, 71% used jets — 41% high viscosity extrusion (HVE), 30% gas dynamic virtual nozzles (GDVN) — 10% used drop-on-tape (DoT), 7% employed fixed targets or chips, 5% SF-ROX and 5% MESH^,. Jet-type techniques are shown in shades of blue. Other includes MESH (~ 5%), free droplets and segmented flow. Conceptually, SF-ROX and MESH can be considered to belong to fixed target and injection sample delivery, respectively. However, both approaches have distinct and unique features: SF-ROX uses very large single crystals and MESH uses high electric fields.

Fig. 4 |. Integration of intensities in rotation- and serial crystallography.

a | The Ewald construction can be used to visualize diffraction geometries. The incoming radiation $\underset{S_0}{\to }$ hits the crystal located at the center of the Ewald sphere, which has radius 1/λ. The reciprocal lattice is drawn, with the origin at the position where the direct beam intersects the Ewald sphere behind the crystal. In conventional rotation crystallography, the crystal is rotated, and the reciprocal lattice rotates (curved arrow), causing reciprocal lattice points (spheres) to move through the Ewald sphere, rotating into and out of reflection condition. b | The observed intensity of diffraction from a certain reciprocal lattice point (such as the red sphere in panel a.) increases, then decreases with rotation angle. Integrating this rocking curve yields the intensity I_hkl. c | In SFX there is no a priori control of the crystal’s orientation, and intersection of the red reciprocal lattice point with the Ewald sphere is a stochastic process. d | The crystals are effectively motionless during the extremely brief exposure. As a result, the observed diffraction corresponds to a thin slice of the rocking curve of each reflection. Partial intensities from many exposures need to be averaged to give I_hkl for each reflection.