Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.
X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.
Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
The self-assembly of strongly-coupled nanocrystal superlattices, as a convenient bottom-up synthesis technique featuring a wide parameter space, is at the forefront of next-generation material design. To realize the full potential of such tunable, functional materials, a more complete understanding of the self-assembly process and the artificial crystals it produces is required. In this work, we discuss the results of a hard coherent X-ray scattering experiment at the Linac Coherent Light Source, observing superlattices long after their initial nucleation. The resulting scattering intensity correlation functions have dispersion suggestive of a disordered crystalline structure and indicate the occurrence of rapid, strain-relieving events therein. We also present real space reconstructions of individual superlattices obtained via coherent diffractive imaging. Through this analysis we thus obtain high-resolution structural and dynamical information of self-assembled superlattices in their native liquid environment.
The XFEL is characterized by high intensity pulses, which are only about 50 femtoseconds in duration. The intensity allows for scattering from microscopic particles, while the short pulses offer a way to outrun radiation damage. XFELs are powerful enough to obliterate most samples in a single pulse. While this allows for a “diffract and destroy” methodology, it also requires instrumentation that can position microscopic particles into the X-ray beam (which may also be microscopic), continuously renew the sample after each pulse, and maintain sample viability during data collection.
Typically these experiments have used liquid microjets to continuously renew sample. The high flow rate associated with liquid microjets requires large amounts of sample, most of which runs to waste between pulses. An injector designed to stream a viscous gel-like material called lipidic cubic phase (LCP) was developed to address this problem. LCP, commonly used as a growth medium for membrane protein crystals, lends itself to low flow rate jetting and so reduces the amount of sample wasted significantly.
This work discusses sample delivery and injection for XFEL experiments. It reviews the liquid microjet method extensively, and presents the LCP injector as a novel device for serial crystallography, including detailed protocols for the LCP injector and anti-settler operation.