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

Instruments / Technical Area	Publication Details	Published
	Morphofunctional changes at the active zone during synaptic vesicle exocytosis Julika Radecke , Raphaela Seeger , Anna Kádková , Ulrike Laugks , Amin Khosrozadeh , Kenneth N. Goldie , Vladan Lučić , Jakob B. Sørensen , Benoît Zuber Embo Reports 2 DOI: 10.15252/embr.202255719 Open Access Show Abstract ▼ Abstract: Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage—late fusion—shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.	Mar 2023
Krios II-Titan Krios II at Diamond	Structural insights into the assembly and activation of the IL ‐27 signaling complex Yibo Jin , Paul K. Fyfe , Scott Gardner , Stephan Wilmes , Doryen Bubeck , Ignacio Moraga Embo Reports DOI: 10.15252/embr.202255450 Diamond Proposal Number(s): [25127] Open Access Show Abstract ▼ Abstract: Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immunosuppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27Rα receptor chains to activate the JAK/STAT signaling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) and AlphaFold modeling to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27Rα (domains 1–2) with GP130 (domains 1–3) to initiate signaling. While both receptors contact the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27Rα and Domain 1 of GP130. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.	Aug 2022
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	The conserved C2 phospholipid‐binding domain in Delta contributes to robust Notch signalling Torcato Martins , Yao Meng , Boguslawa Korona , Richard Suckling , Steven Johnson , Penny A Handford , Susan M. Lea , Sarah J Bray Embo Reports 8 DOI: 10.15252/embr.202152729 Open Access Show Abstract ▼ Abstract: Accurate Notch signalling is critical for development and homeostasis. Fine-tuning of Notch–ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N-terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so-called β1-2 loop that is involved in phospholipid binding. Mutations in the β1-2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the β1-2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss-of-function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine-tuning the balance of trans and cis ligand–receptor interactions.	Aug 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere Julijus Bogomolovas , Jennifer R. Fleming , Barbara Franke , Bruno Manso , Bernd Simon , Alexander Gasch , Marija Markovic , Thomas Brunner , Ralph Knöll , Ju Chen , Siegfried Labeit , Martin Scheffner , Christine Peter , Olga Mayans Embo Reports DOI: 10.15252/embr.201948018 Open Access Show Abstract ▼ Abstract: Striated muscle undergoes remodelling in response to mechanical and physiological stress, but little is known about the integration of such varied signals in the myofibril. The interaction of the elastic kinase region from sarcomeric titin (A168-M1) with the autophagy receptors Nbr1/p62 and MuRF E3 ubiquitin ligases is well suited to link mechanosensing with the trophic response of the myofibril. To investigate the mechanisms of signal cross-talk at this titin node, we elucidated its 3D structure, analysed its response to stretch using steered molecular dynamics simulations and explored its functional relation to MuRF1 and Nbr1/p62 using cellular assays. We found that MuRF1-mediated ubiquitination of titin kinase promotes its scaffolding of Nbr1/p62 and that the process can be dynamically down-regulated by the mechanical unfolding of a linker sequence joining titin kinase with the MuRF1 receptor site in titin. We propose that titin ubiquitination is sensitive to the mechanical state of the sarcomere, the regulation of sarcomere targeting by Nbr1/p62 being a functional outcome. We conclude that MuRF1/Titin Kinase/Nbr1/p62 constitutes a distinct assembly that predictably promotes sarcomere breakdown in inactive muscle.	Aug 2021
I04-Macromolecular Crystallography I23-Long wavelength MX	Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1 Elyse S. Fischer , Conny W. H. Yu , Dom Bellini , Stephen H. Mclaughlin , Christian Orr , Armin Wagner , Stefan M. V. Freund , David Barford Embo Reports 536 DOI: 10.15252/embr.202052242 Open Access Show Abstract ▼ Abstract: During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.	May 2021
Krios IV-Titan Krios IV at Diamond	Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA Martin L. Rennie , Kimon Lemonidis , Connor Arkinson , Viduth K Chaugule , Mairi Clarke , James Streetley , Laura Spagnolo , Helen Walden Embo Reports 21 DOI: 10.15252/embr.202050133 Diamond Proposal Number(s): [16637] Open Access Show Abstract ▼ Abstract: The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double‐stranded DNA via promoting a large‐scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary “Arm” ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1‐UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post‐translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA.	Jul 2020
I04-Macromolecular Crystallography	Mip6 binds directly to the Mex67 UBA domain to maintain low levels of Msn2/4 stress‐dependent mRNAs Manuel Martín‐expósito , Maria‐eugenia Gas , Nada Mohamad , Carme Nuño‐cabanes , Ana Tejada‐colón , Pau Pascual‐garcía , Lorena De La Fuente , Belén Chaves‐arquero , Jonathan Merran , Jeffry Corden , Ana Conesa , José Manuel Pérez‐cañadillas , Jeronimo Bravo , Susana Rodríguez‐navarro Embo Reports 20 DOI: 10.15252/embr.201947964 Diamond Proposal Number(s): [10121] Show Abstract ▼ Abstract: RNA‐binding proteins (RBPs) participate in all steps of gene expression, underscoring their potential as regulators of RNA homeostasis. We structurally and functionally characterize Mip6, a four‐RNA recognition motif (RRM)‐containing RBP, as a functional and physical interactor of the export factor Mex67. Mip6‐RRM4 directly interacts with the ubiquitin‐associated (UBA) domain of Mex67 through a loop containing tryptophan 442. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67‐dependent manner and concentrates in cytoplasmic foci under stress. Photoactivatable ribonucleoside‐enhanced crosslinking and immunoprecipitation experiments show preferential binding of Mip6 to mRNAs regulated by the stress‐response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affects their export and expression levels. Additionally, Mip6 interacts physically and/or functionally with proteins with a role in mRNA metabolism and transcription such as Rrp6, Xrn1, Sgf73, and Rpb1. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4‐dependent transcripts through its direct interaction with the Mex67 UBA domain.	Oct 2019
B21-High Throughput SAXS I04-1-Macromolecular Crystallography (fixed wavelength) Krios I-Titan Krios I at Diamond	The structure of the tetanus toxin reveals pH‐mediated domain dynamics Geoffrey Masuyer , Julian Conrad , Pal Stenmark Embo Reports DOI: 10.15252/embr.201744198 Diamond Proposal Number(s): [11265] Show Abstract ▼ Abstract: The tetanus neurotoxin (TeNT) is a highly potent toxin produced by Clostridium tetani that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal transport. The crystal structure of the tetanus toxin reveals a “closed” domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin's interaction with its polysaccharide receptor. An integrative analysis combining X-ray crystallography, solution scattering and single particle electron cryo-microscopy reveals pH-mediated domain rearrangements that may give TeNT the ability to adapt to the multiple environments encountered during intoxication, and facilitate binding to distinct receptors.	Jun 2017
I02-Macromolecular Crystallography	Control of box C/D snoRNP assembly by N6-methylation of adenine Lin Huang , Saira Ashraf , Jia Wang , David M. J. Lilley Embo Reports DOI: 10.15252/embr.201743967 Diamond Proposal Number(s): [10071, 8268] Open Access Show Abstract ▼ Abstract: N6-methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N6-methyladenine at a key trans Hoogsteen-sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5-kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N6-methylation of adenine prevents the formation of trans Hoogsteen-sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N6mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.	Jun 2017
I03-Macromolecular Crystallography	Structural studies of RFC C tf18 reveal a novel chromatin recruitment role for Dcc1 Benjamin O. Wade , Hon Wing Liu , Catarina P. Samora , Frank Uhlmann , Martin R. Singleton Embo Reports DOI: 10.15252/embr.201642825 Diamond Proposal Number(s): [9826] Open Access Show Abstract ▼ Abstract: Replication factor C complexes load and unload processivity clamps from DNA and are involved in multiple DNA replication and repair pathways. The RFCCtf18 variant complex is required for activation of the intra‐S‐phase checkpoint at stalled replication forks and aids the establishment of sister chromatid cohesion. Unlike other RFC complexes, RFCCtf18 contains two non‐Rfc subunits, Dcc1 and Ctf8. Here, we present the crystal structure of the Dcc1‐Ctf8 heterodimer bound to the C‐terminus of Ctf18. We find that the C‐terminus of Dcc1 contains three‐winged helix domains, which bind to both ssDNA and dsDNA. We further show that these domains are required for full recruitment of the complex to chromatin, and correct activation of the replication checkpoint. These findings provide the first structural data on a eukaryotic seven‐subunit clamp loader and define a new biochemical activity for Dcc1.	Feb 2017

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