Abstract
Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzyme at a high level of detail. The success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.
Highlights
The structural information gathered from X-ray crystallographic studies of proteins is incorporated into many stages of drug development (Congreve et al, 2005; Blundell, 2017)
As a first step in this direction, we investigated chitotriose binding to hen egg-white lysozyme via mix-and-diffuse serial crystallography at a synchrotron source
The serial crystallography experiment was performed on the P11 beamline at PETRA III (Burkhardt et al, 2016) using 13.5 keV photon energy X-rays focused to a spot of 4 Â 8 mm with a flux of 1.6 Â 1013 photons sÀ1
Summary
The structural information gathered from X-ray crystallographic studies of proteins is incorporated into many stages of drug development (Congreve et al, 2005; Blundell, 2017). X-ray crystallographic fragment-screening studies have shown the binding of small molecules that have an affinity that is too low to be detected by chemical assays (Schiebel et al, 2016; Patel et al, 2014; Erlanson et al, 2016). Serial crystallography offers a high-throughput platform to overcome these bottlenecks. This method entails using a Bragg intensity set merged from many snapshot diffraction patterns of individual protein microcrystals to solve the protein structure (Chapman et al, 2011; Boutet et al, 2012). Its use with the brilliant femtosecond pulses of an X-ray free-electron laser has led to a number of GPCR structures (Liu et al, 2013; Zhang et al, 2015; Kang et al, 2015), de novo phasing (Barends et al, 2014; Yamashita et al, 2015; Colletier et al, 2016) and the study of fast light-induced dynamics of photoactive proteins (Tenboer et al, 2014; Barends et al, 2015; Nango et al, 2016; Pande et al, 2016)
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