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Instruments / Technical Area	Publication Details	Published	Actions
I24-Microfocus Macromolecular Crystallography	Enzyme catalysis captured using multiple structures from one crystal at varying temperatures Sam Horrell , Demet Kekilli , Kakali Sen , Robin L. Owen , Florian S. N. Dworkowski , Svetlana V. Antonyuk , Thomas W. Keal , Chin W. Yong , Robert R. Eady , S. Samar Hasnain , Richard W. Strange , Mike Hough Iucrj 5 DOI: 10.1107/S205225251800386X Diamond Proposal Number(s): [11175] Open Access Show Abstract ▼ Abstract: High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate `structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a `top-hat' geometry, which was rapidly transformed to a `side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.	May 2018
I02-Macromolecular Crystallography	Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase Kakali Sen , Sam Horrell , Demet Kekilli , Chin W. Yong , Thomas W. Keal , Hakan Atakisi , David W. Moreau , Robert E. Thorne , Michael A. Hough , Richard W. Strange Iucrj 4 DOI: 10.1107/S2052252517007527 Diamond Proposal Number(s): [13467] Open Access Show Abstract ▼ Abstract: Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (AspCAT and HisCAT) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the AspCAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site `capping residue' (IleCAT), a determinant of ligand binding, are influenced both by temperature and by the protonation state of AspCAT. A previously unobserved conformation of IleCAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.	Jul 2017

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