Sam Horrell

Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal

Serial crystallography has been used to drive copper nitrite reductase through its enzymatic cycle while sampling the same volume of a single cryogenically maintained crystal. A structural movie of X-ray-driven enzyme catalysis has thus been obtained, revealing the structural changes that occur during the catalytic reaction in unprecedented detail.

Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging.

A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material.

Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases

The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation.

Photoreduction and validation of haem-ligand intermediate states in protein crystals by in situ single-crystal spectroscopy and diffraction.

Integrated structural biology can yield powerful synergies and maximize the biological information gained. Two examples are described of combining X-ray crystallography with single-crystal resonance Raman and UV–visible spectroscopies to study the functions of haem proteins.

Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase

Multiple structures obtained from one crystal of copper nitrite reductase at elevated cryogenic temperature, together with molecular-dynamics simulations, reveal catalyically important protein and solvent dynamics at the active site.

MSOX crystallography and simulations to capture redox enzyme catalysis

Michael Alexander Hough1, Demet Kekilli1, Sam Horrell1, Kakali Sen1, Chin Yong2, Thomas W.K. Keal2, Svetlana V Antonyuk3, Robert R. Eady3, S Samar Hasnain3, Richard W. Strange1 1School Of Biological Sciences, Colchester, United Kingdom, 2Scientific Computing Department, STFC Daresbury Laboratory, Warrington, United Kingdom, 3Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom E-mail: mahough@essex.ac.uk

Single Crystal Serial Crystallography to Capture Redox Enzyme Catalysis and Dynamics

Relating individual protein crystal structures to enzyme mechanisms remains a challenging goal for structural biology. The mechanisms of radiation damage to macromolecular crystals have become increasingly well-characterised [1] and attention is paid to minimising the deleterious effects. Alternatively, X-rays may be used to drive enzymes to particular redox states or intermediates. Serial crystallography using multiple crystals has recently been reported in both SR and XFEL experiments [2,3]. I will describe our approach to exploit rapid, shutterless X-ray detector technology on synchrotron MX beamlines to perform low-dose serial crystallography on a single Cu nitrite reductase crystal, allowing 10-50 consecutive X-ray structures at high resolution to be collected, all sampled from the same crystal volume. This serial crystallography approach captures the gradual conversion of the substrate bound at the catalytic type 2 Cu centre, from nitrite to the product, nitric oxide, following reduction of the electron transfer type 1 Cu centre by X-ray generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, which is a vital step in the global denitrification process. We propose that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions in a single protein crystal. It can provide a ‘catalytic reaction movie’ highlighting structural changes that occur during enzyme catalysis. Anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at synchrotron crystallographic beamlines. We describe such serial crystallographic experiments conducted at 100K [4] at the elevated cryogenic temperatures of 180-200K, exploiting previously characterised changes in solvent viscosity and dynamics [5]. Finally, we extend our approach to crystals at room temperature, allowing a more complete protein conformational response to active site structural changes to be observed. References [1] E. F. Garman & M. Weik (2015) J. Synchrotron Rad. 22, 195-200. [2] e.g. M. Suga, et al. (2015) Nature 517, 99-103. [3] e.g. C. Gati, G. et. al. (2014) IUCrJ 1, 87-94. [4] M. Weik & J.-P. Colletier (2010) Acta Cryst. Section D. 66, 437-446. [5] S. Horrell, S. V. et al. (2016) IUCrJ (submitted).