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"The NSF BioXFEL Science and Technology Center (STC) is a new consortium of six research campuses devoted to the application of x-ray free-electron lasers (XFELs) to structural biology. Over the last four years a variety of approaches have been made to the observation of protein structure and dynamics for various classes of proteins. The Linac Coherent Light source at SLAC, the first hard-Xray EXFEL, provides intense coherent hard X-ray pulses at 120 Hz which vaporize protein when focussed to a sub-micron beam. Atomic-resolution Bragg diffraction patterns are nevertheless obtained using 50 fs pulses prior to the onset of significant damage, in this ""diffract-then-destroy"" mode, which outruns radiation damage. This use of short pulses instead of freezing samples to reduce radiation damage therefore opens the way to the study of protein dynamics at room temperature in a native environment. I'll review the work of several groups using a range of approaches to different types of sample, including the following: 1. Differences between the frozen sychrotron structure of GPCR proteins and the RT XFEL structure [1]. 2. Pump-probe dynamic structures in Photosynthesis [2]. 3. XFEL study of 2D protein crystals [3]. 4. Prospects for improved resolution in XFEL imaging from single particles such as viruses, where patterns can be obtained from a single virus. 5. New ideas - the Lipid Cubic Phase injector (which allows protein nanocrystals to be studied also at sychrotrons) [4], prospects for fast Laue diffraction using coherent attosecond X-ray lasers, ab-initio phasing [5], the use of angular correlation functions for analysis of fast solution scattering, and two-color opportunites for serial femtosecond crystallography (SFX). See [6] for a recent review of the field. 1. W.Liu et al Science 342, 1521 (2013) 2. A.Aquila et al Optics Express 20, 2706 (2012) 3. M.Frank et al IUCrJ (2014) In press. 4. U.Weierstall et al Nature Comms. (2014) In press. 5. J. Spence et al Optics Express 19, 2866 (2011). 6. J. Spence et al Rep. Prog. Phys. 75, 102601 (2012)."
Keywords: Biology with X-ray lasers.
abstracts
Membrane proteins are extremely difficult to crystallize, however they are highly important proteins for cellular function. Photosystem I, one of the most complex membrane proteins solved to date took more than a decade to have a structure solved to molecular resolution. Large, well-ordered crystal growth is one of the major bottlenecks in structural determination by x-ray crystallography, due to the difficulty of making the "perfect" crystal. The development of femtosecond nanocrystallography, which uses a stream of fully hydrated nanocrystals to collect diffraction snapshots, effectively reduces this bottleneck[1] Photosystem II changed our biosphere via splitting water and evolving oxygen 2.5 billion years ago. Using femtosecond nanocrystallography we are developing a time-resolved femtosecond crystallography method [2] to unravel the mechanism of water splitting by determining the conformational changes that take place during the oxygen evolution process. Multiple crystallization techniques were originally developed in order to make the nanocrystals necessary for femtosecond nanocrystallography. For Photosystem II nano/microcrystals a free interface diffusion method, is used to increase yield over traditional methods. These crystals are then characterized by three different methods before being used for collecting diff raction data. The three methods currently used are optical microscopy, dynamic light scattering (DLS), and Second Order Nonlinear Imaging of Chiral Crystals (SONICC).
