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Tiit Lukk

The second workshop at the 2015 CHESS Users’ Meeting was organized by MacCHESS scientist Tiit Lukk on the topic of “Fast framing detectors and their applications in time-resolved crystallography/BioSAXS.” The advent of third generation light sources that produce bright X-rays allow scientists to carry out experiments at speeds orders of magnitude faster than was previously possible. This workshop focused on how advances in X-ray detector development have chartered new unexplored avenues into biological sciences, making it possible, for example, to follow enzymatic reactions in real-time as they occur at near atomic resolution.

In the first session Kate Shanks (Gruner detector group, Cornell) spoke of the state of the art of X-ray detector development, highlighting how the field has evolved through time and where the field is headed. She gave an introduction to how fast-framing detectors have benefited the fields of BioSAXS and protein X-ray crystallography in recent years. Robert Thorne (Cornell) gave a presentation on the advances of room temperature structural biology. Thorne reported that 98% of all protein structures are now determined at T=100K, but that crystallographers are starting to realize the value of structural dynamics data that can go missing during sample vitrification (including side chain conformers or subdomain movements). He went on to present evidence for ways to mitigate radiation damage of protein crystals during X-ray diffraction experiments at temperatures between ambient and 100K. Some, but not all, of the damage may be outrun by use of very high dose rates and fast detectors.

Lois Pollack (Cornell) opened the second session, which was dedicated to research and advances in time-resolved BioSAXS (TR-SAXS), a rapidly evolving field enabling scientists to study dynamic biological systems in solution. She presented two contrasting stories utilizing continuous- and stopped-flow set-ups to achieve a time-resolution suitable for determining intermediates in the assembly/disassembly of a nucleosome core particle and the dimerization event of a light-activated LOV domain protein. Tsutomu Matsui (SSRL) added detail to the timeline of TR-SAXS development, pointing out that this powerful technique is still not very well known and only 6% of beamtime at SSRL’s BL4-2 is utilized for TR-SAXS.

Philip Anfinrud (NIH) was the first speaker of the final session on time-resolved crystallography. He discussed recent advances in time-resolved picosecond Laue crystallography and TR-SAXS/WAXS at the BioCARS beamline at the APS. He showed that careful timing of the storage ring bunch spacings, and laser pulses for the activation of the photoactive yellow protein (PYP), enabled the tracking of PYP's reversible photocycle at a 150 picosecond resolution. The experiment enabled Anfinrud and colleagues to assign precise lifetimes to the individual intermediates within the photocycle. Uwe Weierstall (Arizona State University) spoke of recent developments in millisecond serial microcrystallography. Weierstall presented work on a gas shielded gel extrusion device that provides a steady stream of protein microcrystals in a gel-like matrix through a bright X-ray beam. Even though the device was initially designed for an XFEL source, Weierstall demonstrated that the technology can successfully be utilized at a synchrotron source for cases where protein crystals of a few µm in size are available.

The workshop was very well attended and all learned a great deal from the fields’ early adopters and practitioners.

 

 

Submitted by: Tiit Lukk, MacCHESS and Ernest Fontes, CHESS
Cornell University
07/13/2015