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Andriy
Kryshtafovych
,
Reinhard
Albrecht
,
Arnaud
Basle
,
Pedro
Bule
,
Alessandro T.
Caputo
,
Ana Luisa
Carvalho
,
Kinlin L.
Chao
,
Ron
Diskin
,
Krzysztof
Fidelis
,
Carlos M. G. A.
Fontes
,
Folmer
Fredslund
,
Harry J.
Gilbert
,
Celia W.
Goulding
,
Marcus D.
Hartmann
,
Christopher S.
Hayes
,
Osnat
Herzberg
,
Johan C.
Hill
,
Andrzej
Joachimiak
,
Gert-wieland
Kohring
,
Roman I.
Koning
,
Leila
Lo Leggio
,
Marco
Mangiagalli
,
Karolina
Michalska
,
John
Moult
,
Shabir
Najmudin
,
Marco
Nardini
,
Valentina
Nardone
,
Didier
Ndeh
,
Thanh-hong
Nguyen
,
Guido
Pintacuda
,
Sandra
Postel
,
Mark J.
Van Raaij
,
Pietro
Roversi
,
Amir
Shimon
,
Abhimanyu K.
Singh
,
Eric J.
Sundberg
,
Kaspars
Tars
,
Nicole
Zitzmann
,
Torsten
Schwede
Abstract: The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.
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Mar 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
Krios I-Titan Krios I at Diamond
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Pietro
Roversi
,
Lucia
Marti
,
Alessandro T.
Caputo
,
Dominic S.
Alonzi
,
Johan C.
Hill
,
Kyle C.
Dent
,
Abhinav
Kumar
,
Mikail D.
Levasseur
,
Andrea
Lia
,
Thomas
Waksman
,
Souradeep
Basu
,
Yentli Soto
Albrecht
,
Kristin
Qian
,
James Patrick
Mcivor
,
Colette B.
Lipp
,
Dritan
Siliqi
,
Snezana
Vasilijevic
,
Shabaz
Mohammed
,
Petra
Lukacik
,
Martin A.
Walsh
,
Angelo
Santino
,
Nicole
Zitzmann
Abstract: Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.
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Jun 2017
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Jiangyu
Yan
,
Thomas R.
Beattie
,
Adriana L.
Rojas
,
Kelly
Schermerhorn
,
Tamzin
Gristwood
,
Jonathan C.
Trinidad
,
Sonja V.
Albers
,
Pietro
Roversi
,
Andrew F.
Gardner
,
Nicola G. A.
Abrescia
,
Stephen D.
Bell
Diamond Proposal Number(s):
[9063, 10130]
Open Access
Abstract: Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.
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May 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Alessandro T.
Caputo
,
Dominic S.
Alonzi
,
Lucia
Marti
,
Ida-barbara
Reca
,
John
Kiappes
,
Weston B.
Struwe
,
Alice
Cross
,
Souradeep
Basu
,
Ed
Lowe
,
Benoit
Darlot
,
Angelo
Santino
,
Pietro
Roversi
,
Nicole
Zitzmann
Diamond Proposal Number(s):
[9306]
Abstract: The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme’s quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme’s activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.
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Aug 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Verity A.
Jackson
,
Shahid
Mehmood
,
Matthieu
Chavent
,
Pietro
Roversi
,
Maria
Carrasquero
,
Daniel
Del Toro
,
Goenuel
Seyit-bremer
,
Fanomezana M.
Ranaivoson
,
Davide
Comoletti
,
Mark S. P.
Sansom
,
Carol V.
Robinson
,
Rüdiger
Klein
,
Elena
Seiradake
Diamond Proposal Number(s):
[9306, 8423, 1747]
Open Access
Abstract: Latrophilin adhesion-GPCRs (Lphn1–3 or ADGRL1–3) and Unc5 cell guidance receptors (Unc5A–D) interact with FLRT proteins (FLRT1–3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger ‘super-complex’ (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.
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Apr 2016
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I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Jackson et al. describe a crystal structure of mLPHN3 lectin and olfactomedin-like (Olf) domains, revealing the Olf b-propeller fold and calcium-binding site. Assays using HeLa cells and cortical neurons reveal a bi-functional role for Olf and its ligand FLRT, leading to HeLa cell adhesion and neuron repulsion.
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Apr 2015
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Paul
Chappell
,
El Kahina
Meziane
,
Michael
Harrison
,
Łukasz
Magiera
,
Clemens
Hermann
,
Laura
Mears
,
Antoni G
Wrobel
,
Charlotte
Durant
,
Lise Lotte
Nielsen
,
Søren
Buus
,
Nicola
Ternette
,
William
Mwangi
,
Colin
Butter
,
Venugopal
Nair
,
Trudy
Ahyee
,
Richard
Duggleby
,
Alejandro
Madrigal
,
Pietro
Roversi
,
Susan
Lea
,
Jim
Kaufman
Diamond Proposal Number(s):
[9306]
Open Access
Abstract: Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. - See more at: http://elifesciences.org/content/4/e05345#sthash.zKFHdAeD.dpuf
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Apr 2015
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I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[7495, 9306]
Open Access
Abstract: Alkaline phosphatases play a crucial role in phosphate acquisition by microorganisms. To expand our understanding of catalysis by this class of enzymes, we have determined the structure of the widely occurring microbial alkaline phosphatase PhoX. The enzyme contains a complex active-site cofactor comprising two antiferromagnetically coupled ferric iron ions (Fe(3+)), three calcium ions (Ca(2+)), and an oxo group bridging three of the metal ions. Notably, the main part of the cofactor resembles synthetic oxide-centered triangular metal complexes. Structures of PhoX-ligand complexes reveal how the active-site metal ions bind substrate and implicate the cofactor oxo group in the catalytic mechanism. The presence of iron in PhoX raises the possibility that iron bioavailability limits microbial phosphate acquisition.
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Sep 2014
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I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[9306]
Open Access
Abstract: It has recently been shown that the biosynthetic route for both the d1-haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1-haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorinferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorinferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis.
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Apr 2014
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Open Access
Abstract: The collection of articles in this issue forms the proceedings of the 2013 CCP4 Study Weekend Molecular Replacements, which took place on 3–5 January 2013 at the East Midlands Conference Centre of the University of Nottingham, England, UK. As the plural in the chosen title suggests, molecular replacement (MR) these days is relevant in many a field of structural science, well beyond the original task of placing a homologous model in a crystal's unit cell based on its experimentally measured structure-factor amplitudes. In the papers gathered here, MR is cast in contexts as wide and varied as the history of structural science; protein fold and family structural coverage; protein structure modelling; electron microscopy of macromolecular complexes; structural virology; and of course crystallographic software development.
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Nov 2013
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