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54 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
I24-Microfocus Macromolecular Crystallography	Structure of cytosine transport protein CodB provides insight into nucleobase‐cation symporter 1 mechanism Caitlin E. Hatton , Deborah H. Brotherton , Mahalah Spencer , Alexander D Cameron The Embo Journal 19 DOI: 10.15252/embj.2021110527 Open Access Show Abstract ▼ Abstract: CodB is a cytosine transporter from the Nucleobase-Cation-Symport-1 (NCS1) transporter family, a member of the widespread LeuT superfamily. Previous experiments with the nosocomial pathogen Pseudomonas aeruginosa have shown CodB as also important for the uptake of 5-fluorocytosine, which has been suggested as a novel drug to combat antimicrobial resistance by suppressing virulence. Here we solve the crystal structure of CodB from Proteus vulgaris, at 2.4 Å resolution in complex with cytosine. We show that CodB carries out the sodium-dependent uptake of cytosine and can bind 5-fluorocytosine. Comparison of the substrate-bound structures of CodB and the hydantoin transporter Mhp1, the only other NCS1 family member for which the structure is known, highlight the importance of the hydrogen bonds that the substrates make with the main chain at the breakpoint in the discontinuous helix, TM6. In contrast to other LeuT superfamily members, neither CodB nor Mhp1 makes specific interactions with residues on TM1. Comparison of the structures provides insight into the intricate mechanisms of how these proteins transport substrates across the plasma membrane.	Jul 2022
Krios I-Titan Krios I at Diamond	Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase Joseph G. Beton , Jim Monistrol , Anne Wentink , Erin C. Johnston , Anthony J. Roberts , Bernd G. Bukau , Bart W. Hoogenboom , Helen R. Saibil The Embo Journal 9 DOI: 10.15252/embj.2021110410 Diamond Proposal Number(s): [20287] Open Access Show Abstract ▼ Abstract: Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure.	Jun 2022
B21-High Throughput SAXS	Teneurin4 dimer structures reveal a calcium‐stabilized compact conformation supporting homomeric trans‐interactions Dimphna H Meijer , Cátia P Frias , J. Wouter Beugelink , Yanthi N. Deurloo , Bert J. C. Janssen The Embo Journal 66 DOI: 10.15252/embj.2020107505 Diamond Proposal Number(s): [19800] Open Access Show Abstract ▼ Abstract: Establishment of correct synaptic connections is a crucial step during neural circuitry formation. The Teneurin family of neuronal transmembrane proteins promotes cell–cell adhesion via homophilic and heterophilic interactions, and is required for synaptic partner matching in the visual and hippocampal systems in vertebrates. It remains unclear how individual Teneurins form macromolecular cis- and trans-synaptic protein complexes. Here, we present a 2.7 Å cryo-EM structure of the dimeric ectodomain of human Teneurin4. The structure reveals a compact conformation of the dimer, stabilized by interactions mediated by the C-rich, YD-shell, and ABD domains. A 1.5 Å crystal structure of the C-rich domain shows three conserved calcium binding sites, and thermal unfolding assays and SAXS-based rigid-body modeling demonstrate that the compactness and stability of Teneurin4 dimers are calcium-dependent. Teneurin4 dimers form a more extended conformation in conditions that lack calcium. Cellular assays reveal that the compact cis-dimer is compatible with homomeric trans-interactions. Together, these findings support a role for teneurins as a scaffold for macromolecular complex assembly and the establishment of cis- and trans-synaptic interactions to construct functional neuronal circuits.	Jan 2022
Krios IV-Titan Krios IV at Diamond	Helical ordering of envelope‐associated proteins and glycoproteins in respiratory syncytial virus Michaela J Conley , Judith M. Short , Andrew Burns , James Streetley , Joshua Hutchings , Saskia E. Bakker , B. Joanne Power , Hussain Jaffery , Joanne Haney , Giulia Zanetti , Pablo R. Murcia , Murray Stewart , Rachel Fearns , Swetha Vijayakrishnan , David Bhella The Embo Journal 12 DOI: 10.15252/embj.2021109728 Diamond Proposal Number(s): [16637] Open Access Show Abstract ▼ Abstract: Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.	Dec 2021
Krios III-Titan Krios III at Diamond	Cryo‐EM structure of metazoan TRAPPIII, the multi‐subunit complex that activates the GTPase Rab1 Antonio Galindo , Vicente J. Planelles-Herrero , Gianluca Degliesposti , Sean Munro The Embo Journal 40 DOI: 10.15252/embj.2020107608 Diamond Proposal Number(s): [17434] Open Access Show Abstract ▼ Abstract: The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo-EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur-TRAPP.	Jun 2021
Krios III-Titan Krios III at Diamond	Structural basis of TRAPPIII‐mediated Rab1 activation Aaron M. N. Joiner , Ben P. Phillips , Kumar Yugandhar , Ethan J Sanford , Marcus B Smolka , Haiyuan Yu , Elizabeth A. Miller , J Christopher Fromme The Embo Journal 74 DOI: 10.15252/embj.2020107607 Diamond Proposal Number(s): [17434] Open Access Show Abstract ▼ Abstract: The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo-EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α-helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.	May 2021
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	A non‐helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition Dorota Focht , Caroline Neumann , Joseph Lyons , Ander Eguskiza Bilbao , Rickard Blunck , Lina Malinauskaite , Ilona O Schwarz , Jonathan A Javitch , Matthias Quick , Poul Nissen The Embo Journal 40 DOI: 10.15252/embj.2020105164 Show Abstract ▼ Abstract: MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+‐dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate‐bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly‐Met‐Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.	Jan 2021
I24-Microfocus Macromolecular Crystallography	Timeless couples G‐quadruplex detection with processing by DDX 11 helicase during DNA replication Leticia K. Lerner , Sandro Holzer , Mairi Kilkenny , Saša Šviković , Pierre Murat , Davide Schiavone , Cara B Eldridge , Alice Bittleston , Joseph D. Maman , Dana Branzei , Katherine Stott , Luca Pellegrini , Julian E. Sale The Embo Journal 14 DOI: 10.15252/embj.2019104185 Open Access Show Abstract ▼ Abstract: Regions of the genome with the potential to form secondary DNA structures pose a frequent and significant impediment to DNA replication and must be actively managed in order to preserve genetic and epigenetic integrity. How the replisome detects and responds to secondary structures is poorly understood. Here, we show that a core component of the fork protection complex in the eukaryotic replisome, Timeless, harbours in its C‐terminal region a previously unappreciated DNA ‐binding domain that exhibits specific binding to G‐quadruplex (G4) DNA structures. We show that this domain contributes to maintaining processive replication through G4‐forming sequences, and exhibits partial redundancy with an adjacent PARP ‐binding domain. Further, this function of Timeless requires interaction with and activity of the helicase DDX 11. Loss of both Timeless and DDX 11 causes epigenetic instability at G4‐forming sequences and DNA damage. Our findings indicate that Timeless contributes to the ability of the replisome to sense replication‐hindering G4 formation and ensures the prompt resolution of these structures by DDX 11 to maintain processive DNA synthesis.	Jul 2020
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural basis of semaphorin‐plexin cis interaction Daniel Rozbesky , Marieke G. Verhagen , Dimple Karia , Gergely N. Nagy , Luis Alvarez , Ross A Robinson , Karl Harlos , Sergi Padilla‐parra , R. Jeroen Pasterkamp , Edith Y. Jones The Embo Journal 58 DOI: 10.15252/embj.2019102926 Diamond Proposal Number(s): [10627] Open Access Show Abstract ▼ Abstract: Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell‐attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. We have discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. Our high‐resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities.	Jun 2020
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Identification and characterization of diverse OTU deubiquitinases in bacteria Alexander F. Schubert , Justine V Nguyen , Tyler G. Franklin , Paul P. Geurink , Cameron G. Roberts , Daniel J. Sanderson , Lauren N. Miller , Huib Ovaa , Kay Hofmann , Jonathan N. Pruneda , David Komander The Embo Journal 81 DOI: 10.15252/embj.2020105127 Diamond Proposal Number(s): [8547, 11235] Open Access Show Abstract ▼ Abstract: Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, invading pathogens can inactivate immune response pathways and evade detection. The ovarian tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans. Viral pathogens have also extensively co‐opted the OTU fold to subvert host signaling, but the extent to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. By determining the ubiquitin‐bound structures of two examples, we demonstrate the novel strategies that have evolved to both thread an OTU fold and recognize a ubiquitin substrate. With these new examples, we perform the first cross‐kingdom structural analysis of the OTU fold that highlights commonalities among distantly related OTU deubiquitinases.	Jun 2020

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