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

Abstract

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiationsensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β 2 -adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.femtosecond diffraction | crystallography | XFEL | structural biology U sing extremely bright, short-timescale X-ray pulses produced by X-ray free-electron lasers (XFELs), femtosecond crystallography (FX) is an emerging method that expands the structural information accessible from very small or very radiation-sensitive macromolecular crystals. Central to this method is the "diffraction before destruction" (1) process in which a still diffraction image is produced by a single X-ray pulse before significant radiation-induced electronic and atomic rearrangements occur within the crystal (1-3). At the Linac Coherent Light Source (LCLS) at SLAC, a single ∼50-fs-long X-ray pulse can expose a crystal to as many X-ray photons as a typical synchrotron beam line produces in about a second. Exposing small crystals to these intense ultrashort pulses circumvents the dose limitations of conventional X-ray diffraction experiments (4) and may produce useful data to resolutions beyond what is achievable at synchrotrons (5). This innovation provides a pathway to obtain atomic information from proteins that only form micrometer-to nanometer-sized crystals, such as many membrane proteins and large multiprotein complexes. Moreover, XFELs enable "diffraction before reduction" data collection to address another major challenge in structural enzymology by providing a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzyme active sites (6), such as high-valency reaction intermediates that may be significantly photoreduced during a single X-ray exposure at a synchrotron, even at very small doses (7-11). Furthermore, the use of short (tens of femtoseconds) X-ray pulses further complements the structural characterization of biochemical reaction processes by providing access to a time domain two to three orders of magnitude faster (12, 13) than currently accessible using synchrotro...

Crystal structure of the pristine peroxidase ferryl center and its relevance to proton-coupled electron transfer

“…Data were collected at the X-ray pump probe end station at the LCLS in December 2013 and in June 2014 under a stream of liquid nitrogen. Crystals were mounted on a goniometer using the Stanford Automated Mounting System (30), and diffraction patterns were collected on a Rayonix MX325 detector as described previously (31). The helical data collection mode was used in which each crystal was translated and rotated after each exposure (31) so that multiple radiation damage-free diffraction images were obtained from each crystal.…”

Microfluidics: From crystallization to serial time-resolved crystallography

“…One of the simplest approaches to mounting a large number of crystals is the use of traditional loops, 17,184 micromounts, 185 or capillaries 16 to mount a slurry of crystals. 169 It is also possible to mount crystals grown directly on the mount, as in the case of the X-CHIP, where pinned droplets are protected against dehydration by a thin coating of oil (Figure 8(c)) 186,187 or the aforementioned graphene-based microfluidic devices.…”

“…25,31,33,34,37,143,185,[191][192][193][194][195] The simplest mounting strategy involves the deposition of a crystal slurry between two thin silicon nitride wafers. 143 However, most approaches have utilized microfabricated mesh-type structures.…”

“…37 Mesh structures have been fabricated out of silicon 25,31,37,192,193 and polymers such as SU-8 ( Figure 8(d)) 191 and polycarbonate (Figure 8(e)). 185,195 The material of the mesh itself should not affect the quality of the observed diffraction, provided that the crystal is well located within the center of the well. However, care must be taken when crystalline materials such as silicon or metal are used, as intense diffraction from the mesh structure can potentially damage the detector.…”

Crystal Growth of High-Quality Protein Crystals under the Presence of an Alternant Electric Field in Pulse-Wave Mode, and a Strong Magnetic Field with Radio Frequency Pulses Characterized by X-ray Diffraction

“…That is why just a few 3D structures using neutron diffraction have been deposited in the Protein Data Bank (PDB). Despite these apparent disadvantages, there have been recent significant advances, not only in the beam lines and the detectors technology, but also in the sample preparation (perdeuteration), though the second via to obtain the 3D structure from nanocrystals is the free-electron lasers to perform X-ray crystallography of macromolecular complexes [6][7][8][9][10][11][12]. Additionally, magnetic fields [13][14][15][16][17][18], electric fields [16,19,20] or a combination of both [21] have proven to be efficient in obtaining bigger and higher-quality protein single crystals for X-ray crystallography.…”