Featured Technology

Delivering on the Promise of Free Electron Lasers

Though a relatively new technology to the field of structural biology, the free electron laser shows great promise for providing insight into the function of large biomolecules and complexes. The first X-ray free electron laser (XFEL), the Linac Coherent Light Source (LCLS), was developed at the SLAC National Accelerator Laboratory and started operation in 2009. Currently, there are several others in operation or being planned around the world, mainly in Europe and Japan.

Initial XFEL experiments were performed by a team under the leadership of John Spence, SpenceRichard Snell Professor of Physics at Arizona State University, which included Petra Fromme, Paul V. Galvin Professor of Chemistry & Biochemistry at Arizona State University, and the Principal Investigator of the Protein Structure Initiative Center for 3PCQ Membrane Proteins in Infectious Diseases (MPID). In this experiment, single crystal x-ray diffraction ‘snapshots’ were collected from fully a hydrated stream of membrane protein complex photosystem I nanocrystals. The beam is so powerful that it damages the crystal as soon as the pulse from the XFEL hits the sample. In this technique, called serial femtosecond X-ray crystallography (SFX), the length of the pulse is kept very short to allow for diffraction before the crystal is destroyed. The data from nanocrystals of the thermophilic cyanobacterium Synechococcus elongates Photosystem I resulted in an interpretable 8.5-Å resolution electron density map and resulted in the structure shown on the left, 3PCQ (Chapman, et al., 2011).

The following year, Spence and Fromme were involved in a study that established the feasibility of using a free electron laser to study crystals grown in lipidic sponge phase. 4AC5 Employing a sponge phase micro-jet and SFX, Johannson, et al. (2012) recorded interpretable diffraction data for the Blastochloris viridis photosynthetic reaction center. Using these data, they were able to solve the structure to 8.2-Å resolution using molecular replacement (4AC5, pictured right).

Since these inaugural experiments, novel tools and applications have continued to be developed, expanding the power of this technique. Two program suites, CrystFEL (White, et al., 2013) and cctbx.xfel (Sauter, et al., 2013), have been developed to analyze the large amounts of data that are generated over the course of these experiments. 4NC3 Recently, researchers from the PSI Center for Membrane Protein Structure Determination GPCR Network used a cubic lipidic phase jet and SFX to solve the structure of a human serotonin 5-HT2B receptor bound to the agonist ergotamine (4NC3, left). The microcrystals studied averaged about 125 µm3 in size, and were previously unsuitable for use in traditional diffraction experiments (Liu, et al., 2013).

Several groups have extended the reach of this method, such as by determining protein structures using XFEL-obtained de novo phasing (Barends, et al., 2013) and combining spectroscopy with SFX to determine the catalytic cycle of Photosystem II (PSII; Kern, et al., 2012, Kern et al., 2014, Kupitz et al., 2014). FEL users uniquely are able to perform X-ray emission spectroscopy to detect a change in a metal charge density while simultaneously conducting a diffraction experiment. In this case, it enables researchers to see the progression of the Mn4Ca complex, which sits at the heart of the oxygen evolving center of PSII, as it cycles through five states to split water into O2, protons, and electrons. Aside from advancing efforts to visualize biological processes occurring in real time on an atomic scale, information from the study of PSII could assist in the development of solar-energy based water-splitting devices to affect artificial photosynthesis (Read more at redOrbit).

Last year, the use of FELs for biological applications got a vote of confidence from the National Science Foundation (NSF). BioXFEL logo The University at Buffalo (UB) in New York was awarded a grant that helped launch the NSF Science and Technology Center for Biology with X-ray Free Electron Lasers, or BioXFEL. This new interdisciplinary research center will focus on exploring new bio-imaging techniques, particularly for observing molecular machines, using the immensely powerful X-ray free electron laser technology. BioXFEL, under the direction of Eaton E. Lattman, Professor of Structural Biology and Chief Executive Officer of the Hauptman-Woodward Medical Research Institute (HWI), researchers will use XFEL to conduct a wide range of scientific inquiry, including studies of photosynthesis, enzyme function at the atomic level, and drug-delivery proteins. During this exciting time in the field of structural and functional biology, it is important that centers like BioXFEL have as part of their mission to educate and provide new tools and training about this technique to the wider scientific community. This dedication will allow researchers to develop this method and realize its potential to capture biomolecular machines in action.

Read more about the PSI GPCR structure in the SBKB April 2014 Technical Highlight, Membrane Proteome: Microcrystals Yield Big Data.