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55 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength)	Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR Niko Amin-wetzel , Lisa Neidhardt , Yahui Yan , Matthias P. Mayer , David Ron Elife 8 DOI: 10.7554/eLife.50793 Diamond Proposal Number(s): [15916] Show Abstract ▼ Abstract: Coupling of endoplasmic reticulum stress to dimerisation‑dependent activation of the UPR transducer IRE1 is incompletely understood. Whilst the luminal co-chaperone ERdj4 promotes a complex between the Hsp70 BiP and IRE1's stress-sensing luminal domain (IRE1LD) that favours the latter's monomeric inactive state and loss of ERdj4 de-represses IRE1, evidence linking these cellular and in vitro observations is presently lacking. We report that enforced loading of endogenous BiP onto endogenous IRE1α repressed UPR signalling in CHO cells and deletions in the IRE1α locus that de-repressed the UPR in cells, encode flexible regions of IRE1LD that mediated BiP‑induced monomerisation in vitro. Changes in the hydrogen exchange mass spectrometry profile of IRE1LD induced by ERdj4 and BiP confirmed monomerisation and were consistent with active destabilisation of the IRE1LD dimer. Together, these observations support a competition model whereby waning ER stress passively partitions ERdj4 and BiP to IRE1LD to initiate active repression of UPR signalling.	Dec 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin Cassandra L. Stowe , Thomas A. Burley , Helen Allan , Maria Vinci , Gabriela Kramer-marek , Daniela M Ciobota , Gary N. Parkinson , Tara L. Southworth , Giulia Agliardi , Alastair Hotblack , Mark F. Lythgoe , Bruce R. Branchini , Tammy L. Kalber , James C Anderson , Martin A. Pule Elife 8 DOI: 10.7554/eLife.45801 Show Abstract ▼ Abstract: Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH2) analogue infraluciferin (iLH2), near-infrared dual BLI can be achievedin vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5'-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH2 reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH2 is superior to LH2 for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model.	Oct 2019
I03-Macromolecular Crystallography	Protein engineering expands the effector recognition profile of a rice NLR immune receptor Juan Carlos De La Concepcion , Marina Franceschetti , Dan Maclean , Ryohei Terauchi , Sophien Kamoun , Mark J. Banfield Elife 8 DOI: 10.7554/eLife.47713 Diamond Proposal Number(s): [13467] Open Access Show Abstract ▼ Abstract: Plant nucleotide binding, leucine-rich repeat (NLR) receptors detect pathogen effectors and initiate an immune response. Since their discovery, NLRs have been the focus of protein engineering to improve disease resistance. However, this approach has proven challenging, in part due to their narrow response specificity. Previously, we revealed the structural basis of pathogen recognition by the integrated heavy metal associated (HMA) domain of the rice NLR Pikp (Maqbool et al., 2015). Here, we used structure-guided engineering to expand the response profile of Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector-binding interface of the integrated Pikp–HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell-death response to AVR-Pik variants previously unrecognized by Pikp in planta. The structures of the engineered Pikp–HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly where this interaction occurs via integrated domains.	Sep 2019
I03-Macromolecular Crystallography	Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B Timo Vögtle , Sumana Sharma , Jun Mori , Zoltan Nagy , Daniela Semeniak , Cyril Scandola , Mitchell J. Geer , Christopher W. Smith , Jordan Lane , Scott Pollack , Riitta Lassila , Annukka Jouppila , Alastair J. Barr , Derek J. Ogg , Tina D. Howard , Helen J. Mcmiken , Juli Warwicker , Catherine Geh , Rachel Rowlinson , W. Mark Abbott , Anita Eckly , Harald Schulze , Gavin J. Wright , Alexandra Mazharian , Klaus Futterer , Sundaresan Rajesh , Michael R. Douglas , Yotis A. Senis Elife 8 DOI: 10.7554/eLife.46840 Diamond Proposal Number(s): [20026] Open Access Show Abstract ▼ Abstract: The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically-modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.	Aug 2019
Krios I-Titan Krios I at Diamond	Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule Samuel E. Lacey , Shaoda He , Sjors H. W. Scheres , Andrew P. Carter Elife 8 DOI: 10.7554/eLife.47145 Diamond Proposal Number(s): [17434] Open Access Show Abstract ▼ Abstract: Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of axonemes in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a coiled-coil stalk. We address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using helical Relion. The majority of the MTBD is rigid upon binding, with the transition to the high-affinity state controlled by the movement of a single helix at the MTBD interface. DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that contacts the adjacent protofilament. Unexpectedly, we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.	Jul 2019
Krios II-Titan Krios II at Diamond	Mechanism of completion of peptidyltransferase centre assembly in eukaryotes Vasileios Kargas , Pablo Castro-hartmann , Norberto Escudero-urquijo , Kyle Dent , Christine Hilcenko , Carolin Sailer , Gertrude Zisser , Maria J. Marques-carvalho , Simone Pellegrino , Leszek Wawiórka , Stefan M. V. Freund , Jane L. Wagstaff , Antonina Andreeva , Alexandre Faille , Edwin Chen , Florian Stengel , Helmut Bergler , Alan John Warren Elife 8 DOI: 10.7554/eLife.44904 Diamond Proposal Number(s): [14359, 15368, 16940, 17057, 18078, 18328] Show Abstract ▼ Abstract: During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.	May 2019
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Phosphorylation-mediated interactions with TOPBP1 couple 53BP1 and 9-1-1 to control the G1 DNA damage checkpoint Nicolas Bigot , Matthew Day , Robert A. Baldock , Felicity Z. Watts , Antony W. Oliver , Laurence H. Pearl Elife DOI: 10.7554/eLife.44353 Diamond Proposal Number(s): [10088] Open Access Show Abstract ▼ Abstract: Coordination of the cellular response to DNA damage is organised by multi-domain 'scaffold' proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint.	May 2019
I13-2-Diamond Manchester Imaging	Bumblebee visual allometry results in locally improved resolution and globally improved sensitivity Gavin J. Taylor , Pierre Tichit , Marie D. Schmidt , Andrew J. Bodey , Christoph Rau , Emily Baird Elife 8 DOI: 10.7554/eLife.40613 Diamond Proposal Number(s): [13848, 16052] Open Access Show Abstract ▼ Abstract: The quality of visual information that is available to an animal is limited by the size of its eyes. Differences in eye size can be observed even between closely related individuals, yet we understand little about how this affects vision. Insects are good models for exploring the effects of size on visual systems because many insect species exhibit size polymorphism. Previous work has been limited by difficulties in determining the 3D structure of eyes. We have developed a novel method based on x-ray microtomography to measure the 3D structure of insect eyes and to calculate predictions of their visual capabilities. We used our method to investigate visual allometry in the bumblebee Bombus terrestris and found that size affects specific aspects of vision, including binocular overlap, optical sensitivity, and dorsofrontal visual resolution. This reveals that differential scaling between eye areas provides flexibility that improves the visual capabilities of larger bumblebees.	Feb 2019
Krios I-Titan Krios I at Diamond	Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer’s and Pick’s diseases Wenjuan Zhang , Benjamin Falcon , Alexey G. Murzin , Juan Fan , R. Anthony Crowther , Michel Goedert , Sjors H. W. Scheres Elife 8 DOI: 10.7554/eLife.43584 Diamond Proposal Number(s): [17434] Open Access Show Abstract ▼ Abstract: Assembly of microtubule-associated protein tau into filamentous inclusions underlies a range of neurodegenerative diseases. Tau filaments adopt different conformations in Alzheimer’s and Pick’s diseases. Here, we used cryo- and immuno- electron microscopy to characterise filaments that were assembled from recombinant full-length human tau with four (2N4R) or three (2N3R) microtubule-binding repeats in the presence of heparin. 2N4R tau assembles into multiple types of filaments, and the structures of three types reveal similar ‘kinked hairpin’ folds, in which the second and third repeats pack against each other. 2N3R tau filaments are structurally homogeneous, and adopt a dimeric core, where the third repeats of two tau molecules pack in a parallel manner. The heparin-induced tau filaments differ from those of Alzheimer’s or Pick’s disease, which have larger cores with different repeat compositions. Our results illustrate the structural versatility of amyloid filaments, and raise questions about the relevance of in vitro assembly.	Feb 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Preacinetobactin not acinetobactin is essential for iron uptake by the BauA transporter of the pathogen Acinetobacter baumannii Lucile Moynie , Ilaria Serra , Mariano A. Scorciapino , Emilia Oueis , Malcolm G. P. Page , Matteo Ceccarelli , James Naismith Elife 7 DOI: 10.7554/eLife.42270 Open Access Show Abstract ▼ Abstract: New strategies are urgently required to develop antibiotics. The siderophore uptake system has attracted considerable attention but rational design of siderophore antibiotic conjugates requires knowledge of recognition by the cognate outer membrane transporter. Acinetobacter baumannii is a serious pathogen, which utilizes (pre)acinetobactin to scavenge iron from the host. We report the structure of the (pre)acinetobactin transporter BauA bound to the siderophore, identifying the structural determinants of recognition. Detailed biophysical analysis confirms that BauA recognises preacinetobactin. We show that acinetobactin is not recognised by the protein thus preacinetobactin is essential for iron uptake. The structure shows and NMR confirms that under physiological conditions a molecule of acinetobactin will bind to two free coordination sites on the iron preacinetobactin complex. The ability to recognise a heterotrimeric iron preacinetobactin acinetobactin complex may rationalize contradictory reports in the literature. These results open new avenues for the design of novel antibiotic conjugates (trojan horse) antibiotics.	Dec 2018

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