Monday, January 5th 2015
Femtosecond crystallography (FX) is especially suitable for studying radiation sensitive enzymes that require metals for their function, as the extremely short and bright X-ray pulses can produce a diffraction image before any atomic motions can occur in the crystal. This cutting edge method is capable of extending our capacity to study smaller, more fragile crystals and determine the catalytic structures of biologically relevant macromolecules.
In conventional X-ray crystallography experiments, one crystal is mounted on a goniometer, which is then used to rotate the sample in the X-ray beam. The short and bright X-ray pulses produced by the free-electron laser (XFEL) at the SLAC National Acceleratory Laboratory’s Linac Coherent Light Source (LCLS) damage or destroy crystals nearly instantaneously, requiring tens of thousands of crystals to be used in experiments. Recently, researchers in the Physical Biosciences Division (PBD) of the Lawrence Berkeley National Laboratory (Berkeley Lab) collaborated on a technique that will extend the ability of scientists to perform efficient and flexible FX experiments.
Currently, most FX researchers use injectors to deliver a continuous stream of crystals to the beam, which wastes a large portion of material. Alternate methods are being developed to expose a single drop of the crystalline material to the beam at one time, but these have not been perfected. In an article published on December 2, 2014 in PNAS, lead author Aina Cohen of SLAC describes an experiment using a goniometer-based method to mount samples in the path of the FEL. James Holton, PBD Biophysicist Faculty Scientist and the director of Beamline 8.3.1 at the Advanced Light Source (ALS), assisted in designing the experiment, which utilized equipment that was developed previously at SLAC’s LCLS and Stanford Synchrotron Radiation Light Source (SSRL). “Ever since the Braggs did their original X-ray crystallography work in 1914, crystals have been rotated during data collection to smooth over a myriad of difficulties. Now that XFEL pulses are far too fast to do this, Dr. Cohen and I had to return to these first principles in designing the data collection protocol,” Holton said. The resulting highly automated system uses specialized sample containers and customized software, allowing for efficient data collection and decreased crystalline sample consumption.
To compare the utility of this setup, researchers collected data from two types of protein crystals (two macromolecular complexes and two metalloenzymes) using both goniometer- and injector-based delivery. Nicholas Sauter, Computational Staff Scientist in PBD, along with two postdoctoral researchers in his group, Aaron Brewster and Johan Hattne (now a Research Specialist at Janelia Farm Research Campus), were part of the team that analyzed these data. “Most of the FX experiments were done with a moving stream of crystals, but that made it hard to collect and process data,” said Sauter. “Goniometer methods are familiar to all crystallographers. The fact that they work with femtosecond X-ray pulses makes this technique accessible to everyone’s protein crystals.” cctbx.xfel, open-source software for free-electron laser data processing previously developed by Sauter and his group, was used during the experiment for quasi-real time data analysis.
Aside from SLAC and Berkeley Lab, Cohen’s research team was made up of participants from the Stanford University School of Medicine, the University of Pittsburgh School of Medicine, Howard Hughes Medical Institute, Montana State University, and the University of California, San Francisco. Employing automated goniometer-based instrumentation allowed researchers to determine high-resolution structures of four molecules using FX on just a fraction of the crystals typically required using injector-based methods. “In doing this, we sparked a revolution in how we think about X-ray diffraction data all around the world, and processing software has been improving by leaps and bounds ever since,” said Holton. “The original protocol we developed is already obsolete, and soon even synchrotron-based data collection may follow suit, taking advantage of the fundamentally superior data quality from non-rotating crystals for the first time in 100 years.” By reducing both crystalline material sample and data processing requirements, the team has effectively decreased the overall cost and time needed to perform these cutting-edge scientific experiments and has taken steps that will eventually lead to wider use of this technology.