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100 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6, 7, 8 [Next/Last]

Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond	Shigella flexneri evades LPS ubiquitylation through IpaH1.4-mediated degradation of RNF213 Katerina Naydenova , Keith B. Boyle , Claudio Pathe , Prathyush Pothukuchi , Ana Crespillo-Casado , Felix Scharte , Pierre-Mehdi Hammoudi , Elsje G. Otten , Neal M. Alto , Felix Randow Nature Structural & Molecular Biology 62 DOI: 10.1038/s41594-025-01530-8 Diamond Proposal Number(s): [31336] Open Access Show Abstract ▼ Abstract: Pathogens have evolved diverse strategies to counteract host immunity. Ubiquitylation of lipopolysaccharide (LPS) on cytosol-invading bacteria by the E3 ligase RNF213 creates ‘eat me’ signals for antibacterial autophagy, but whether and how cytosol-adapted bacteria avoid LPS ubiquitylation remains poorly understood. Here, we show that the enterobacterium Shigella flexneri actively antagonizes LPS ubiquitylation through IpaH1.4, a secreted effector protein with ubiquitin E3 ligase activity. IpaH1.4 binds to RNF213, ubiquitylates it and targets it for proteasomal degradation, thus counteracting host-protective LPS ubiquitylation. To understand how IpaH1.4 recognizes RNF213, we determined the cryogenic electron microscopy structure of the IpaH1.4–RNF213 complex. The specificity of the interaction is achieved through the leucine-rich repeat of IpaH1.4, which binds the RING domain of RNF213 by hijacking the conserved RING interface required for binding to ubiquitin-charged E2 enzymes. IpaH1.4 also targets other E3 ligases involved in inflammation and immunity through binding to the E2-interacting face of their RING domains, including the E3 ligase LUBAC that is required for the synthesis of M1-linked ubiquitin chains on cytosol-invading bacteria downstream of RNF213. We conclude that IpaH1.4 has evolved to antagonize multiple antibacterial and proinflammatory host E3 ligases.	Apr 2025
I04-Macromolecular Crystallography	Capture, mutual inhibition and release mechanism for aPKC–Par6 and its multisite polarity substrate Lgl Christopher P. Earl , Mathias Cobbaut , André Barros-Carvalho , Marina E. Ivanova , David C. Briggs , Eurico Morais-De-Sá , Peter J. Parker , Neil Q. Mcdonald Nature Structural & Molecular Biology 23 DOI: 10.1038/s41594-024-01425-0 Open Access Show Abstract ▼ Abstract: The mutually antagonistic relationship of atypical protein kinase C (aPKC) and partitioning-defective protein 6 (Par6) with the substrate lethal (2) giant larvae (Lgl) is essential for regulating polarity across many cell types. Although aPKC–Par6 phosphorylates Lgl at three serine sites to exclude it from the apical domain, aPKC–Par6 and Lgl paradoxically form a stable kinase–substrate complex, with conflicting roles proposed for Par6. We report the structure of human aPKCι–Par6α bound to full-length Llgl1, captured through an aPKCι docking site and a Par6PDZ contact. This complex traps a phospho-S663 Llgl1 intermediate bridging between aPKC and Par6, impeding phosphorylation progression. Thus, aPKCι is effectively inhibited by Llgl1pS663 while Llgl1 is captured by aPKCι–Par6. Mutational disruption of the Lgl–aPKC interaction impedes complex assembly and Lgl phosphorylation, whereas disrupting the Lgl–Par6PDZ contact promotes complex dissociation and Lgl phosphorylation. We demonstrate a Par6PDZ-regulated substrate capture-and-release model requiring binding by active Cdc42 and the apical partner Crumbs to drive complex disassembly. Our results suggest a mechanism for mutual regulation and spatial control of aPKC–Par6 and Lgl activities.	Jan 2025
Krios I-Titan Krios I at Diamond	Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair Souvik Naskar , Andrea Merino , Javier Espadas , Jayanti Singh , Aurelien Roux , Adai Colom , Harry H. Low Nature Structural & Molecular Biology 184 DOI: 10.1038/s41594-024-01401-8 Open Access Show Abstract ▼ Abstract: The ESCRT-III-like protein Vipp1 couples filament polymerization with membrane remodeling. It assembles planar sheets as well as 3D rings and helical polymers, all implicated in mitigating plastid-associated membrane stress. The architecture of Vipp1 planar sheets and helical polymers remains unknown, as do the geometric changes required to transition between polymeric forms. Here we show how cyanobacterial Vipp1 assembles into morphologically-related sheets and spirals on membranes in vitro. The spirals converge to form a central ring similar to those described in membrane budding. Cryo-EM structures of helical filaments reveal a close geometric relationship between Vipp1 helical and planar lattices. Moreover, the helical structures reveal how filaments twist—a process required for Vipp1, and likely other ESCRT-III filaments, to transition between planar and 3D architectures. Overall, our results provide a molecular model for Vipp1 ring biogenesis and a mechanism for Vipp1 membrane stabilization and repair, with implications for other ESCRT-III systems.	Nov 2024
Krios I-Titan Krios I at Diamond	Structural basis for activity switching in polymerases determining the fate of let-7 pre-miRNAs Gangshun Yi , Mingda Ye , Loic Carrique , Afaf H. El-Sagheer , Tom Brown , Chris J. Norbury , Peijun Zhang , Robert J. C. Gilbert Nature Structural & Molecular Biology 20 DOI: 10.1038/s41594-024-01357-9 Diamond Proposal Number(s): [20223, 21004] Open Access Show Abstract ▼ Abstract: Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3′ end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A. Our structures reveal that pre-miRNAs engage the enzymes in fundamentally different ways depending on the presence of LIN28A, which clamps them onto the TUTs to enable processive 3′ oligo-uridylation. This study reveals the molecular basis for mono- versus oligo-uridylation by TUT7/4, as determined by the presence of LIN28A, and thus their mechanism of action in the regulation of cell fate and in cancer.	Jul 2024
I03-Macromolecular Crystallography	Molecular stripping underpins derepression of a toxin–antitoxin system Grzegorz J. Grabe , Rachel T. Giorgio , Miłosz Wieczór , Bridget Gollan , Molly Sargen , Modesto Orozco , Stephen A. Hare , Sophie Helaine Nature Structural & Molecular Biology 181 DOI: 10.1038/s41594-024-01253-2 Diamond Proposal Number(s): [17221] Show Abstract ▼ Abstract: Transcription factors control gene expression; among these, transcriptional repressors must liberate the promoter for derepression to occur. Toxin–antitoxin (TA) modules are bacterial elements that autoregulate their transcription by binding the promoter in a T:A ratio-dependent manner, known as conditional cooperativity. The molecular basis of how excess toxin triggers derepression has remained elusive, largely because monitoring the rearrangement of promoter–repressor complexes, which underpin derepression, is challenging. Here, we dissect the autoregulation of the Salmonella enterica tacAT3 module. Using a combination of assays targeting DNA binding and promoter activity, as well as structural characterization, we determine the essential TA and DNA elements required to control transcription, and we reconstitute a repression-to-derepression path. We demonstrate that excess toxin triggers molecular stripping of the repressor complex off the DNA through multiple allosteric changes causing DNA distortion and ultimately leading to derepression. Thus, our work provides important insight into the mechanisms underlying conditional cooperativity.	Mar 2024
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	UBE2A and UBE2B are recruited by an atypical E3 ligase module in UBR4 Lucy Barnsby-Greer , Peter D. Mabbitt , Marc-Andre Dery , Daniel R. Squair , Nicola T. Wood , Frederic Lamoliatte , Sven M. Lange , Satpal Virdee Nature Structural & Molecular Biology 116 DOI: 10.1038/s41594-023-01192-4 Open Access Show Abstract ▼ Abstract: UBR4 is a 574 kDa E3 ligase (E3) of the N-degron pathway with roles in neurodevelopment, age-associated muscular atrophy and cancer. The catalytic module that carries out ubiquitin (Ub) transfer remains unknown. Here we identify and characterize a distinct E3 module within human UBR4 consisting of a ‘hemiRING’ zinc finger, a helical-rich UBR zinc-finger interacting (UZI) subdomain, and an N-terminal region that can serve as an affinity factor for the E2 conjugating enzyme (E2). The structure of an E2–E3 complex provides atomic-level insight into the specificity determinants of the hemiRING toward the cognate E2s UBE2A/UBE2B. Via an allosteric mechanism, the UZI subdomain modestly activates the Ub-loaded E2 (E2∼Ub). We propose attenuated activation is complemented by the intrinsically high lysine reactivity of UBE2A, and their cooperation imparts a reactivity profile important for substrate specificity and optimal degradation kinetics. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains with Ub E3 activity.	Jan 2024
I04-Macromolecular Crystallography	The molecular basis of drug selectivity for α5 subunit-containing GABAA receptors Vikram Babu Kasaragod , Tomas Malinauskas , Ayla A. Wahid , Judith Lengyel , Frederic Knoflach , Steven W. Hardwick , Charlotte F. Jones , Wan-Na Chen , Xavier Lucas , Kamel El Omari , Dimitri Y. Chirgadze , A. Radu Aricescu , Giuseppe Cecere , Maria-Clemencia Hernandez , Paul S. Miller Nature Structural & Molecular Biology 90 DOI: 10.1038/s41594-023-01133-1 Diamond Proposal Number(s): [10627] Open Access Show Abstract ▼ Abstract: α5 subunit-containing γ-aminobutyric acid type A (GABAA) receptors represent a promising drug target for neurological and neuropsychiatric disorders. Altered expression and function contributes to neurodevelopmental disorders such as Dup15q and Angelman syndromes, developmental epilepsy and autism. Effective drug action without side effects is dependent on both α5-subtype selectivity and the strength of the positive or negative allosteric modulation (PAM or NAM). Here we solve structures of drugs bound to the α5 subunit. These define the molecular basis of binding and α5 selectivity of the β-carboline, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), type II benzodiazepine NAMs, and a series of isoxazole NAMs and PAMs. For the isoxazole series, each molecule appears as an ‘upper’ and ‘lower’ moiety in the pocket. Structural data and radioligand binding data reveal a positional displacement of the upper moiety containing the isoxazole between the NAMs and PAMs. Using a hybrid molecule we directly measure the functional contribution of the upper moiety to NAM versus PAM activity. Overall, these structures provide a framework by which to understand distinct modulator binding modes and their basis of α5-subtype selectivity, appreciate structure–activity relationships, and empower future structure-based drug design campaigns.	Oct 2023
Krios IV-Titan Krios IV at Diamond	Specific recognition and ubiquitination of translating ribosomes by mammalian CCR4–NOT Eva Absmeier , Viswanathan Chandrasekaran , Francis J. O'Reilly , James A. W. Stowell , Juri Rappsilber , Lori Passmore Nature Structural & Molecular Biology 11 DOI: 10.1038/s41594-023-01075-8 Diamond Proposal Number(s): [23268] Show Abstract ▼ Abstract: Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4–Not deadenylation complex. The details of this process in mammals remain unclear. Here, we use cryogenic electron microscopy (cryo-EM) and crosslinking mass spectrometry to show that mammalian CCR4–NOT specifically recognizes ribosomes that are stalled during translation elongation in an in vitro reconstituted system with rabbit and human components. Similar to yeast, mammalian CCR4–NOT inserts a helical bundle of its CNOT3 subunit into the empty E site of the ribosome. Our cryo-EM structure shows that CNOT3 also locks the L1 stalk in an open conformation to inhibit further translation. CCR4–NOT is required for stable association of the nonconstitutive subunit CNOT4, which ubiquitinates the ribosome, likely to signal stalled translation elongation. Overall, our work shows that human CCR4–NOT not only detects but also enforces ribosomal stalling to couple translation and mRNA decay.	Aug 2023
Krios I-Titan Krios I at Diamond	Cryo-EM structure of GABA transporter 1 reveals substrate recognition and transport mechanism Smruti Ranjan Nayak , Deepthi Joseph , Georg Höfner , Archishman Dakua , Arunabh Athreya , Klaus T. Wanner , Baruch I. Kanner , Aravind Penmatsa Nature Structural & Molecular Biology 187 DOI: 10.1038/s41594-023-01011-w Diamond Proposal Number(s): [31643] Open Access Show Abstract ▼ Abstract: The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.	Jul 2023
Krios I-Titan Krios I at Diamond	Architecture of the ESCPE-1 membrane coat Carlos Lopez-Robles , Stefano Scaramuzza , Elsa Astorga-Simon , Morié Ishida , Chad D. Williamson , Soledad Banos-Mateos , David Gil-Carton , Miguel Romero-Durana , Ander Vidaurrazaga , Juan Fernandez-Recio , Adriana L. Rojas , Juan S. Bonifacino , Daniel Castaño-Díez , Aitor Hierro Nature Structural & Molecular Biology 23 DOI: 10.1038/s41594-023-01014-7 Diamond Proposal Number(s): [20113, 17171] Open Access Show Abstract ▼ Abstract: Recycling of membrane proteins enables the reuse of receptors, ion channels and transporters. A key component of the recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), which rescues transmembrane proteins from the endolysosomal pathway for transport to the trans-Golgi network and the plasma membrane. This rescue entails the formation of recycling tubules through ESCPE-1 recruitment, cargo capture, coat assembly and membrane sculpting by mechanisms that remain largely unknown. Herein, we show that ESCPE-1 has a single-layer coat organization and suggest how synergistic interactions between ESCPE-1 protomers, phosphoinositides and cargo molecules result in a global arrangement of amphipathic helices to drive tubule formation. Our results thus define a key process of tubule-based endosomal sorting.	Jun 2023

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