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30 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	PK1 from Drosophila obscurin is an inactive pseudokinase with scaffolding properties Thomas Zacharchenko , Till Dorendorf , Nicolas Locker , Evert Van Dijk , Anja Katzemich , Kay Diederichs , Belinda Bullard , Olga Mayans Open Biology 13 DOI: 10.1098/rsob.220350 Diamond Proposal Number(s): [8997] Open Access Show Abstract ▼ Abstract: Obscurins are large filamentous proteins with crucial roles in the assembly, stability and regulation of muscle. Characteristic of these proteins is a tandem of two C-terminal kinase domains, PK1 and PK2, that are separated by a long intrinsically disordered sequence. The significance of this conserved domain arrangement is unknown. Our study of PK1 from Drosophila obscurin shows that this is a pseudokinase with features typical of the CAM-kinase family, but which carries a minimalistic regulatory tail that no longer binds calmodulin or has mechanosensory properties typical of other sarcomeric kinases. PK1 binds ATP with high affinity, but in the absence of magnesium and lacks detectable phosphotransfer activity. It also has a highly diverged active site, strictly conserved across arthropods, that might have evolved to accommodate an unconventional binder. We find that PK1 interacts with PK2, suggesting a functional relation to the latter. These findings lead us to speculate that PK1/PK2 form a pseudokinase/kinase dual system, where PK1 might act as an allosteric regulator of PK2 and where mechanosensing properties, akin to those described for regulatory tails in titin-like kinases, might now reside on the unstructured interkinase segment. We propose that the PK1-interkinase-PK2 region constitutes an integrated functional unit in obscurin proteins.	Apr 2023
Krios I-Titan Krios I at Diamond	The lipid linked oligosaccharide polymerase Wzy and its regulating co-polymerase, Wzz, from enterobacterial common antigen biosynthesis form a complex Miriam Weckener , Laura S. Woodward , Bradley R. Clarke , Huanting Liu , Philip N. Ward , Audrey Le Bas , David Bhella , Chris Whitfield , James H. Naismith Open Biology 13 DOI: 10.1098/rsob.220373 Diamond Proposal Number(s): [16637] Open Access Show Abstract ▼ Abstract: The enterobacterial common antigen (ECA) is a carbohydrate polymer that is associated with the cell envelope in the Enterobacteriaceae. ECA contains a repeating trisaccharide which is polymerized by WzyE, a member of the Wzy membrane protein polymerase superfamily. WzyE activity is regulated by a membrane protein polysaccharide co-polymerase, WzzE. Förster resonance energy transfer experiments demonstrate that WzyE and WzzE from Pectobacterium atrosepticum form a complex in vivo, and immunoblotting and cryo-electron microscopy (cryo-EM) analysis confirm a defined stoichiometry of approximately eight WzzE to one WzyE. Low-resolution cryo-EM reconstructions of the complex, aided by an antibody recognizing the C-terminus of WzyE, reveals WzyE sits in the central membrane lumen formed by the octameric arrangement of the transmembrane helices of WzzE. The pairing of Wzy and Wzz is found in polymerization systems for other bacterial polymers, including lipopolysaccharide O-antigens and capsular polysaccharides. The data provide new structural insight into a conserved mechanism for regulating polysaccharide chain length in bacteria.	Mar 2023
I03-Macromolecular Crystallography	Experimental and computational snapshots of C-C bond formation in a C-nucleoside synthase Wenbo Li , Georgina C. Girt , Ashish Radadiya , James J. P. Stewart , Nigel G. J. Richards , James H. Naismith Open Biology 13 DOI: 10.1098/rsob.220287 Open Access Show Abstract ▼ Abstract: The biosynthetic enzyme, ForT, catalyses the formation of a C-C bond between 4-amino-1H-pyrazoledicarboxylic acid and MgPRPP to produce a C-nucleoside precursor of formycin A. The transformation catalysed by ForT is of chemical interest because it is one of only a few examples in which C-C bond formation takes place via an electrophilic substitution of a small, aromatic heterocycle. In addition, ForT is capable of discriminating between the aminopyrazoledicarboxylic acid and an analogue in which the amine is replaced by a hydroxyl group; a remarkable feat given the steric and electronic similarities of the two molecules. Here we report biophysical measurements, structural biology and quantum chemical calculations that provide a detailed molecular picture of ForT-catalysed C-C bond formation and the conformational changes that are coupled to catalysis. Our findings set the scene for employing engineered ForT variants in the biocatalytic production of novel, anti-viral C-nucleoside and C-nucleotide analogues.	Jan 2023
I03-Macromolecular Crystallography	The zinc-binding motif in tankyrases is required for the structural integrity of the catalytic ADP-ribosyltransferase domain Sven T. Sowa , Lari Lehtio Open Biology 12 DOI: 10.1098/rsob.210365 Diamond Proposal Number(s): [19951] Open Access Show Abstract ▼ Abstract: Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on the activity, stability and structure of human tankyrases. We generated mutants of human tankyrase (TNKS) 1 and TNKS2, abolishing the zinc-binding capabilities, and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive, probably due to introduction of major structural defects, the TNKS2 mutant remained active and displayed an altered activity profile compared to the wild-type.	Mar 2022
I03-Macromolecular Crystallography	Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism Nehad Noby , Husam Sabah Auhim , Samuel Winter , Harley L. Worthy , Amira M. Embaby , Hesham Saeed , Ahmed Hussein , Christopher R. Pudney , Pierre J. Rizkallah , Stephen A. Wells , D. Dafydd Jones Open Biology 11 DOI: 10.1098/rsob.210182 Diamond Proposal Number(s): [18812] Open Access Show Abstract ▼ Abstract: Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.	Dec 2021
	Parakeet: a digital twin software pipeline to assess the impact of experimental parameters on tomographic reconstructions for cryo-electron tomography James M. Parkhurst , Maud Dumoux , Mark Basham , Daniel Clare , C. Alistair Siebert , Trond Varslot , Angus Kirkland , James H. Naismith , Gwyndaf Evans Open Biology 11 DOI: 10.1098/rsob.210160 Open Access Show Abstract ▼ Abstract: In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a ‘digital twin’ software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.	Oct 2021
I04-Macromolecular Crystallography	Bdellovibrio bacteriovorus phosphoglucose isomerase structures reveal novel rigidity in the active site of a selected subset of enzymes upon substrate binding R. W. Meek , I. T. Cadby , A. L. Lovering Open Biology 11 DOI: 10.1098/rsob.210098 Diamond Proposal Number(s): [10369, 14692] Open Access Show Abstract ▼ Abstract: Glycolysis and gluconeogenesis are central pathways of metabolism across all domains of life. A prominent enzyme in these pathways is phosphoglucose isomerase (PGI), which mediates the interconversion of glucose-6-phosphate and fructose-6-phosphate. The predatory bacterium Bdellovibrio bacteriovorus leads a complex life cycle, switching between intraperiplasmic replicative and extracellular ‘hunter’ attack-phase stages. Passage through this complex life cycle involves different metabolic states. Here we present the unliganded and substrate-bound structures of the B. bacteriovorus PGI, solved to 1.74 Å and 1.67 Å, respectively. These structures reveal that an induced-fit conformational change within the active site is not a prerequisite for the binding of substrates in some PGIs. Crucially, we suggest a phenylalanine residue, conserved across most PGI enzymes but substituted for glycine in B. bacteriovorus and other select organisms, is central to the induced-fit mode of substrate recognition for PGIs. This enzyme also represents the smallest conventional PGI characterized to date and probably represents the minimal requirements for a functional PGI.	Aug 2021
B16-Test Beamline B21-High Throughput SAXS	Structural basis for the coiled-coil architecture of human CtIP C. R. Morton , N. J. Rzechorzek , J. D. Maman , M. Kuramochi , H. Sekiguchi , R. Rambo , Y. C. Sasaki , O. R. Davies , Luca Pellegrini Open Biology 11 DOI: 10.1098/rsob.210060 Open Access Show Abstract ▼ Abstract: The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.	Jun 2021
I03-Macromolecular Crystallography	Viral macrodomains: a structural and evolutionary assessment of the pharmacological potential Johannes Gregor Matthias Rack , Valentina Zorzini , Zihan Zhu , Marion Schuller , Dragana Ahel , Ivan Ahel Open Biology 10 DOI: 10.1098/rsob.200237 Diamond Proposal Number(s): [27001] Open Access Show Abstract ▼ Abstract: Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.	Nov 2020
I02-Macromolecular Crystallography	The efficiency of insulin production and its content in insulin-expressing model β-cells correlate with their Zn 2+ levels Petra Dzianová , Seiya Asai , Martina Chrudinová , Lucie Kosinová , Pavlo Potalitsyn , Pavel Šácha , Romana Hadravová , Irena Selicharová , Jan Kříž , Johan P. Turkenburg , Andrzej Marek Brzozowski , Jiří Jiráček , Lenka Žáková Open Biology 10 DOI: 10.1098/rsob.200137 Diamond Proposal Number(s): [13587] Open Access Show Abstract ▼ Abstract: Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.	Oct 2020

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