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22 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Crystal structure of the [2Fe–2S] protein I (Shethna protein I) from Azotobacter vinelandii Burak V. Kabasakal , Charles A. R. Cotton , James W. Murray Acta Crystallographica Section F Structural Biology Communications 77, 407 - 411 DOI: 10.1107/S2053230X21009936 Diamond Proposal Number(s): [9424] Open Access Show Abstract ▼ Abstract: Azotobacter vinelandii is a model diazotroph and is the source of most nitrogenase material for structural and biochemical work. Azotobacter can grow in above-atmospheric levels of oxygen, despite the sensitivity of nitrogenase activity to oxygen. Azotobacter has many iron–sulfur proteins in its genome, which were identified as far back as the 1960s and probably play roles in the complex redox chemistry that Azotobacter must maintain when fixing nitrogen. Here, the 2.1 Å resolution crystal structure of the [2Fe–2S] protein I (Shethna protein I) from A. vinelandii is presented, revealing a homodimer with the [2Fe–2S] cluster coordinated by the surrounding conserved cysteine residues. It is similar to the structure of the thioredoxin-like [2Fe–2S] protein from Aquifex aeolicus, including the positions of the [2Fe–2S] clusters and conserved cysteine residues. The structure of Shethna protein I will provide information for understanding its function in relation to nitrogen fixation and its evolutionary relationships to other ferredoxins.	Nov 2021
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Versatile selective evolutionary pressure using synthetic defect in universal metabolism Lara Sellés Vidal , James W. Murray , John T. Heap Nature Communications 12 DOI: 10.1038/s41467-021-27266-9 Diamond Proposal Number(s): [12579] Open Access Show Abstract ▼ Abstract: The non-natural needs of industrial applications often require new or improved enzymes. The structures and properties of enzymes are difficult to predict or design de novo. Instead, semi-rational approaches mimicking evolution entail diversification of parent enzymes followed by evaluation of isolated variants. Artificial selection pressures coupling desired enzyme properties to cell growth could overcome this key bottleneck, but are usually narrow in scope. Here we show diverse enzymes using the ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) can substitute for defective NAD regeneration, representing a very broadly-applicable artificial selection. Inactivation of Escherichia coli genes required for anaerobic NAD regeneration causes a conditional growth defect. Cells are rescued by foreign enzymes connected to the metabolic network only via NAD or NADP, but only when their substrates are supplied. Using this principle, alcohol dehydrogenase, imine reductase and nitroreductase variants with desired selectivity modifications, and a high-performing isopropanol metabolic pathway, are isolated from libraries of millions of variants in single-round experiments with typical limited information to guide design.	Nov 2021
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Structure-guided discovery of potent and selective DYRK1A inhibitors Csaba Weber , Melinda Sipos , Attila Paczal , Balazs Balint , Vilibald Kun , Nicolas Foloppe , Pawel Dokurno , Andrew J. Massey , David Lee Walmsley , Roderick E. Hubbard , James Murray , Karen Benwell , Thomas Edmonds , Didier Demarles , Alain Bruno , Mike Burbridge , Francisco Cruzalegui , Andras Kotschy Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.1c00023 Diamond Proposal Number(s): [1857, 2103] Show Abstract ▼ Abstract: The kinase DYRK1A is an attractive target for drug discovery programs due to its implication in multiple diseases. Through a fragment screen, we identified a simple biaryl compound that is bound to the DYRK1A ATP site with very high efficiency, although with limited selectivity. Structure-guided optimization cycles enabled us to convert this fragment hit into potent and selective DYRK1A inhibitors. Exploiting the structural differences in DYRK1A and its close homologue DYRK2, we were able to fine-tune the selectivity of our inhibitors. Our best compounds potently inhibited DYRK1A in the cell culture and in vivo and demonstrated drug-like properties. The inhibition of DYRK1A in vivo translated into dose-dependent tumor growth inhibition in a model of ovarian carcinoma.	May 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	Rapid optimisation of fragments and hits to lead compounds from screening of crude reaction mixtures Lisa M. Baker , Anthony Aimon , James B. Murray , Allan E. Surgenor , Natalia Matassova , Stephen D. Roughley , Patrick M. Collins , Tobias Krojer , Frank Von Delft , Roderick E. Hubbard Communications Chemistry 3 DOI: 10.1038/s42004-020-00367-0 Open Access Show Abstract ▼ Abstract: Fragment based methods are now widely used to identify starting points in drug discovery and generation of tools for chemical biology. A significant challenge is optimization of these weak binding fragments to hit and lead compounds. We have developed an approach where individual reaction mixtures of analogues of hits can be evaluated without purification of the product. Here, we describe experiments to optimise the processes and then assess such mixtures in the high throughput crystal structure determination facility, XChem. Diffraction data for crystals of the proteins Hsp90 and PDHK2 soaked individually with 83 crude reaction mixtures are analysed manually or with the automated XChem procedures. The results of structural analysis are compared with binding measurements from other biophysical techniques. This approach can transform early hit to lead optimisation and the lessons learnt from this study provide a protocol that can be used by the community.	Sep 2020
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography Krios I-Titan Krios I at Diamond Krios III-Titan Krios III at Diamond	Structural basis of light-induced redox regulation in the Calvin–Benson cycle in cyanobacteria Ciaran R. Mcfarlane , Nita R. Shah , Burak V. Kabasakal , Blanca Echeverria , Charles Cotton , Doryen Bubeck , James W. Murray Proceedings Of The National Academy Of Sciences 75 DOI: 10.1073/pnas.1906722116 Diamond Proposal Number(s): [19432, 18659, 12579] Open Access Show Abstract ▼ Abstract: Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin–Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.	Sep 2019
I03-Macromolecular Crystallography	A low-potential terminal oxidase associated with the iron-only nitrogenase from the nitrogen-fixing bacterium Azotobacter vinelandii Febin Varghese , Burak Veli Kabasakal , Charles A. R. Cotton , Jörg Schumacher , A. William Rutherford , Andrea Fantuzzi , James W. Murray Journal Of Biological Chemistry DOI: 10.1074/jbc.RA118.007285 Diamond Proposal Number(s): [12579] Show Abstract ▼ Abstract: The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the flavin adenine dinucleotide cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 mV and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.	May 2019
I02-Macromolecular Crystallography	Coping with strong translational noncrystallographic symmetry and extreme anisotropy in molecular replacement with Phaser : human Rab27a Mostafa Jamshidiha , Inmaculada Pérez-Dorado , James W. Murray , Edward W. Tate , Ernesto Cota , Randy J. Read Acta Crystallographica Section D Structural Biology 75, 342 - 353 DOI: 10.1107/S2059798318017825 Diamond Proposal Number(s): [9424] Open Access Show Abstract ▼ Abstract: Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27aMut, crystallized within 24 h and diffracted to 2.82 Å resolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by Phaser were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in Phaser. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in Phaser, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more suitable crystal forms.	Mar 2019
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein Jianfeng Yu , Jana Knoppová , Franck Michoux , Wojciech Bialek , Ernesto Cota , Mahendra K. Shukla , Adéla Strašková , Guillem Pascual Aznar , Roman Sobotka , Josef Komenda , James W. Murray , Peter J. Nixon Proceedings Of The National Academy Of Sciences 49 DOI: 10.1073/pnas.1800609115 Diamond Proposal Number(s): [7299, 12579] Show Abstract ▼ Abstract: Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved “Arg patch” on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.	Jul 2018
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structure and interactions of the TPR domain of Sgt2 with yeast chaperones and Ybr137wp Ewelina M. Krysztofinska , Nicola J. Evans , Arjun Thapaliya , James Murray , Rhodri M. L. Morgan , Santiago Martinez-Lumbreras , Rivka L. Isaacson Frontiers In Molecular Biosciences 4 DOI: 10.3389/fmolb.2017.00068 Diamond Proposal Number(s): [12579, 13597] Open Access Show Abstract ▼ Abstract: Small glutamine-rich tetratricopeptide repeat-containing protein 2 (Sgt2) is a multi-module co-chaperone involved in several protein quality control pathways. The TPR domain of Sgt2 and several other proteins, including SGTA, Hop, and CHIP, is a highly conserved motif known to form transient complexes with molecular chaperones such as Hsp70 and Hsp90. In this work, we present the first high resolution crystal structures of Sgt2_TPR alone and in complex with a C-terminal peptide PTVEEVD from heat shock protein, Ssa1. Using nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, we demonstrate that Sgt2_TPR interacts with peptides corresponding to the C-termini of Ssa1, Hsc82, and Ybr137wp with similar binding modes and affinities.	Oct 2017
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Structural basis for the mechanism of ATP-dependent acetone carboxylation Florence Mus , Brian J. Eilers , Alexander B. Alleman , Burak V. Kabasakal , Jennifer N. Wells , James W. Murray , Boguslaw P. Nocek , Jennifer L. Dubois , John W. Peters Scientific Reports 7 DOI: 10.1038/s41598-017-06973-8 Diamond Proposal Number(s): [9424, 12579] Open Access Show Abstract ▼ Abstract: Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon dioxide gas (CO2) or its hydrated form, bicarbonate (HCO3−), into target molecules. Acetone carboxylases (ACs) catalyze the conversion of substrates acetone and HCO3− to form the product acetoacetate. Many bicarbonate-incorporating carboxylases rely on the organic cofactor biotin for the activation of bicarbonate. ACs contain metal ions but not organic cofactors, and use ATP to activate substrates through phosphorylation. How the enzyme coordinates these phosphorylation events and new C-C bond formation in the absence of biotin has remained a mystery since these enzymes were discovered. The first structural rationale for acetone carboxylation is presented here, focusing on the 360 kDa (αβγ)2 heterohexameric AC from Xanthobacter autotrophicus in the ligand-free, AMP-bound, and acetate coordinated states. These structures suggest successive steps in a catalytic cycle revealing that AC undergoes large conformational changes coupled to substrate activation by ATP to perform C-C bond ligation at a distant Mn center. These results illustrate a new chemical strategy for the conversion of CO2 into biomass, a process of great significance to the global carbon cycle.	Aug 2017

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