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

Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography Krios I-Titan Krios I 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
I04-1-Macromolecular Crystallography (fixed wavelength)	Structure of Psb29/Thf1 and its association with the FtsH protease complex involved in photosystem II repair in cyanobacteria Martina Bec̆ková , Jianfeng Yu , Vendula Krynická , Amanda Kozlo , Shengxi Shao , Peter Koník , Josef Komenda , James W. Murray , Peter J. Nixon Philosophical Transactions Of The Royal Society B: Biological Sciences 372 DOI: 10.1098/rstb.2016.0394 Diamond Proposal Number(s): [7299] Open Access Show Abstract ▼ Abstract: One strategy for enhancing photosynthesis in crop plants is to improve their ability to repair photosystem II (PSII) in response to irreversible damage by light. Despite the pivotal role of thylakoid-embedded FtsH protease complexes in the selective degradation of PSII subunits during repair, little is known about the factors involved in regulating FtsH expression. Here we show using the cyanobacterium Synechocystis sp. PCC 6803 that the Psb29 subunit, originally identified as a minor component of His-tagged PSII preparations, physically interacts with FtsH complexes in vivo and is required for normal accumulation of the FtsH2/FtsH3 hetero-oligomeric complex involved in PSII repair. We show using X-ray crystallography that Psb29 from Thermosynechococcus elongatus has a unique fold consisting of a helical bundle and an extended C-terminal helix and contains a highly conserved region that might be involved in binding to FtsH. A similar interaction is likely to occur in Arabidopsis chloroplasts between the Psb29 homologue, termed THF1, and the FTSH2/FTSH5 complex. The direct involvement of Psb29/THF1 in FtsH accumulation helps explain why THF1 is a target during the hypersensitive response in plants induced by pathogen infection. Downregulating FtsH function and the PSII repair cycle via THF1 would contribute to the production of reactive oxygen species, the loss of chloroplast function and cell death.	Aug 2017
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension James T. Macdonald , Burak V. Kabasakal , David Godding , Sebastian Kraatz , Louie Henderson , James Barber , Paul Freemont , James W. Murray Proceedings Of The National Academy Of Sciences 113, 10346 - 10351 DOI: 10.1073/pnas.1525308113 Diamond Proposal Number(s): [7299, 1227, 9424] Open Access Show Abstract ▼ Abstract: The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computationally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto a synthetic protein core without the use of backbone fragments from known structures. Two other loop designs were not clearly resolved in the crystal structures, and one loop appeared to be in an incorrect conformation. We have also shown that the repeat unit can accommodate whole-domain insertions by inserting a domain into one of the designed loops.	Sep 2016
I02-Macromolecular Crystallography	Identification Of The Elusive Pyruvate Reductase Of Chlamydomonas reinhardtii Chloroplasts S. J. Burgess , H. Taha , J. A. Yeoman , O. Iamshanova , K. X. Chan , M. Boehm , J. Bundy , W. Bialek , J. W. Murray , P. J. Nixon Plant And Cell Physiology DOI: 10.1093/pcp/pcv167 PMID: 26574578 Diamond Proposal Number(s): [7299] Open Access Show Abstract ▼ Abstract: Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including D-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their sub-cellular locations; however, the identity of the enzyme involved in reducing pyruvate to D-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants we conclude that pyruvate reduction in the chloroplast is catalysed by a tetrameric NAD+-dependent D-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a 'lactate valve' for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent D-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm.	Nov 2015
I04-Macromolecular Crystallography	Structure of the Dual-Function Fructose-1,6/Sedoheptulose-1, 7-Bisphosphatase from Thermosynechococcus Elongatus Bound with Sedoheptulose-7-Phosphate Charles Cotton , Burak Kabasakal , Nishat A. Miah , James W Murray Acta Crystallographica Section F Structural Biology Communications 71, 1341 - 1345 DOI: 10.1107/S2053230X15016829 PMID: 26457528 Diamond Proposal Number(s): [7299] Show Abstract ▼ Abstract: The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria carries out two activities in the Calvin cycle. Structures of this enzyme from the cyanobacterium Synechocystis sp. PCC 6803 exist, but only with adenosine monophosphate (AMP) or fructose-1,6-bisphosphate and AMP bound. The mechanisms which control both selectivity between the two sugars and the structural mechanisms for redox control are still unresolved. Here, the structure of the dual-function FBP/SBPase from the thermophilic cyanobacterium Thermosynechococcus elongatus is presented with sedoheptulose-7-phosphate bound and in the absence of AMP. The structure is globally very similar to the Synechocystis sp. PCC 6803 enzyme, but highlights features of selectivity at the active site and loop ordering at the AMP-binding site. Understanding the selectivity and control of this enzyme is critical for understanding the Calvin cycle in cyanobacteria and for possible biotechnological application in plants.	Oct 2015

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