Abstract
Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth ('pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments. The structures of proteinase K (PK) and A2A adenosine receptor (A2AAR) were determined to resolutions of 1.8 and 4.2 Å using 4 and 24 consecutive 100 ps X-ray pulse exposures, respectively. Strong PK data were processed using existing Laue approaches, while weaker A2AAR data required an alternative data-processing strategy. This demonstration of the feasibility presents new opportunities for time-resolved experiments with microcrystals to study structural changes in real time at pink-beam synchrotron beamlines worldwide.
Highlights
In recent years, the field of structural biology has experienced numerous technological breakthroughs that have accelerated protein structure determination
Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs), in which diffraction snapshots are collected from thousands of nano- or microcrystals in random orientations (Chapman et al, 2011), has solved two major technical challenges of conventional synchrotron singlecrystal methods: (1) the need for production of large, well diffracting crystals and (2) inevitable radiation damage associated with X-ray exposures
We used an lipidic cubic phase (LCP) injector designed by Uwe Weierstall at Arizona State University (Weierstall et al, 2014) to deliver microcrystals of a size range between 5 mm (A2AAR) and 10–15 mm (PK)
Summary
The field of structural biology has experienced numerous technological breakthroughs that have accelerated protein structure determination Among these advances are high-brightness photon sources such as X-ray free-electron lasers (XFELs), fast read-out detectors (e.g. CSPAD, AGIPD and EIGER) and novel sample-delivery methods (e.g. electrospun liquid microjets, microfluidic devices, GDVN injector and high-viscosity injector) (Ayvazyan et al, 2006; Feld et al, 2015; Huang et al, 2015; Sierra et al, 2012; Weierstall et al, 2014, 2012). Microfocus beamlines optimized for macromolecular crystallography at third-generation synchrotrons can produce highly brilliant (up to 1013 photons sÀ1) and highly focused (as small as 1 mm) X-ray beams They are equipped with fastreadout detectors (e.g. PILATUS and EIGER), which have enabled a rapid expansion of the SMX approach to many synchrotron facilities, demonstrating its advantages compared with traditional single-crystal crystallography. It has been shown that room-temperature SMX experiments at synchrotrons can outrun secondary radiation damage (Owen et al, 2012; Warkentin et al, 2013, 2017)
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