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72 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	FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair Pablo Alcon , Shabih Shakeel , Zhuo A. Chen , Juri Rappsilber , Ketan J. Patel , Lori Passmore Nature Structural & Molecular Biology 493 DOI: 10.1038/s41594-020-0380-1 Diamond Proposal Number(s): [17434, 23268] Show Abstract ▼ Abstract: Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2–FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2–FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2–FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.	Feb 2020
Krios I-Titan Krios I at Diamond Krios II-Titan Krios II at Diamond Krios IV-Titan Krios IV at Diamond	A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair Kaiying Cheng , Martin Wilkinson , Yuriy Chaban , Dale B. Wigley Nature Structural & Molecular Biology 27, 71 - 77 DOI: 10.1038/s41594-019-0355-2 Diamond Proposal Number(s): [19865] Open Access Show Abstract ▼ Abstract: The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We report cryo-EM structures of the Escherichia coli RecBCD complex bound to several different DNA forks containing a Chi sequence, including one in which Chi is recognized and others in which it is not. The Chi-recognized structure shows conformational changes in regions of the protein that contact Chi and reveals a tortuous path taken by the DNA. Sequence specificity arises from interactions with both the RecC subunit and the sequence itself. These structures provide molecular details for how Chi is recognized and insights into the changes that occur in response to Chi binding that switch RecBCD from bacteriophage destruction and CRISPR spacer acquisition to constructive host DNA repair.	Jan 2020
Krios III-Titan Krios III at Diamond	Mechanism of ribosome stalling during translation of a poly(A) tail Viswanathan Chandrasekaran , Szymon Juszkiewicz , Junhong Choi , Joseph D. Puglisi , Alan Brown , Sichen Shao , V. Ramakrishnan , Ramanujan S. Hegde Nature Structural & Molecular Biology 26, 1132 - 1140 DOI: 10.1038/s41594-019-0331-x Diamond Proposal Number(s): [17434] Show Abstract ▼ Abstract: Faulty or damaged messenger RNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein degradation and ribosome recycling. The most common mRNA defect in eukaryotes is probably inappropriate polyadenylation at near-cognate sites within the coding region. How ribosomes stall selectively when they encounter poly(A) is unclear. Here, we use biochemical and structural approaches in mammalian systems to show that poly-lysine, encoded by poly(A), favors a peptidyl-transfer RNA conformation suboptimal for peptide bond formation. This conformation partially slows elongation, permitting poly(A) mRNA in the ribosome’s decoding center to adopt a ribosomal RNA-stabilized single-stranded helix. The reconfigured decoding center clashes with incoming aminoacyl-tRNA, thereby precluding elongation. Thus, coincidence detection of poly-lysine in the exit tunnel and poly(A) in the decoding center allows ribosomes to detect aberrant mRNAs selectively, stall elongation and trigger downstream quality control pathways essential for cellular homeostasis.	Nov 2019
Krios I-Titan Krios I at Diamond Krios II-Titan Krios II at Diamond	Structure of the dynein-2 complex and its assembly with intraflagellar transport trains Katerina Toropova , Ruta Zalyte , Aakash G. Mukhopadhyay , Miroslav Mladenov , Andrew Carter , Anthony J. Roberts Nature Structural & Molecular Biology 26, 823 - 829 DOI: 10.1038/s41594-019-0286-y Diamond Proposal Number(s): [14704] Show Abstract ▼ Abstract: Dynein-2 assembles with polymeric intraflagellar transport (IFT) trains to form a transport machinery that is crucial for cilia biogenesis and signaling. Here we recombinantly expressed the ~1.4-MDa human dynein-2 complex and solved its cryo-EM structure to near-atomic resolution. The two identical copies of the dynein-2 heavy chain are contorted into different conformations by a WDR60−WDR34 heterodimer and a block of two RB and six LC8 light chains. One heavy chain is steered into a zig-zag conformation, which matches the periodicity of the anterograde IFT-B train. Contacts between adjacent dyneins along the train indicate a cooperative mode of assembly. Removal of the WDR60−WDR34−light chain subcomplex renders dynein-2 monomeric and relieves autoinhibition of its motility. Our results converge on a model in which an unusual stoichiometry of non-motor subunits controls dynein-2 assembly, asymmetry, and activity, giving mechanistic insight into the interaction of dynein-2 with IFT trains and the origin of diverse functions in the dynein family.	Aug 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	The intrinsic structure of poly(A) RNA determines the specificity of Pan2 and Caf1 deadenylases Terence T. L. Tang , James A. W. Stowell , Chris H. Hill , Lori Passmore Nature Structural & Molecular Biology 2 DOI: 10.1038/s41594-019-0227-9 Diamond Proposal Number(s): [15916] Show Abstract ▼ Abstract: The 3′ poly(A) tail of messenger RNA is fundamental to regulating eukaryotic gene expression. Shortening of the poly(A) tail, termed deadenylation, reduces transcript stability and inhibits translation. Nonetheless, the mechanism for poly(A) recognition by the conserved deadenylase complexes Pan2–Pan3 and Ccr4–Not is poorly understood. Here we provide a model for poly(A) RNA recognition by two DEDD-family deadenylase enzymes, Pan2 and the Ccr4–Not nuclease Caf1. Crystal structures of Saccharomyces cerevisiae Pan2 in complex with RNA show that, surprisingly, Pan2 does not form canonical base-specific contacts. Instead, it recognizes the intrinsic stacked, helical conformation of poly(A) RNA. Using a fully reconstituted biochemical system, we show that disruption of this structure—for example, by incorporation of guanosine into poly(A)—inhibits deadenylation by both Pan2 and Caf1. Together, these data establish a paradigm for specific recognition of the conformation of poly(A) RNA by proteins that regulate gene expression.	May 2019
	Structural basis for the delivery of activated sialic acid into Golgi for sialyation Emmanuel Nji , Ashutosh Gulati , Abdul Aziz Qureshi , Mathieu Coincon , David Drew Nature Structural & Molecular Biology 14 DOI: 10.1038/s41594-019-0225-y Show Abstract ▼ Abstract: The decoration of secretory glycoproteins and glycolipids with sialic acid is critical to many physiological and pathological processes. Sialyation is dependent on a continuous supply of sialic acid into Golgi organelles in the form of CMP-sialic acid. Translocation of CMP-sialic acid into Golgi is carried out by the CMP-sialic acid transporter (CST). Mutations in human CST are linked to glycosylation disorders, and CST is important for glycopathway engineering, as it is critical for sialyation efficiency of therapeutic glycoproteins. The mechanism of how CMP-sialic acid is recognized and translocated across Golgi membranes in exchange for CMP is poorly understood. Here we have determined the crystal structure of a Zea mays CST in complex with CMP. We conclude that the specificity of CST for CMP-sialic acid is established by the recognition of the nucleotide CMP to such an extent that they are mechanistically capable of both passive and coupled antiporter activity.	May 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	A folded conformation of MukBEF and cohesin Frank Bürmann , Byung-gil Lee , Thane Than , Ludwig Sinn , Francis J. O'reilly , Stanislau Yatskevich , Juri Rappsilber , Bin Hu , Kim Nasmyth , Jan Lowe Nature Structural & Molecular Biology 26, 227 - 236 DOI: 10.1038/s41594-019-0196-z Diamond Proposal Number(s): [15916] Show Abstract ▼ Abstract: Structural maintenance of chromosomes (SMC)–kleisin complexes organize chromosomal DNAs in all domains of life, with key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through the entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC–kleisin complexes: MukBEF from Escherichia coli, and cohesin from Saccharomyces cerevisiae. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head–kleisin module, situated at opposite ends of the arms. Our findings favour SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA contact sites during DNA loading and translocation.	Mar 2019
Krios II-Titan Krios II at Diamond	Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc1 Andrew M. Hartley , Natalya Lukoyanova , Yunyi Zhang , Alfredo Cabrera-orefice , Susanne Arnold , Brigitte Meunier , Nikos Pinotsis , Amandine Marechal Nature Structural & Molecular Biology 26, 78 - 83 DOI: 10.1038/s41594-018-0172-z Diamond Proposal Number(s): [14704] Show Abstract ▼ Abstract: Cytochrome c oxidase (complex IV, CIV) is known in mammals to exist independently or in association with other respiratory proteins to form supercomplexes (SCs). In Saccharomyces cerevisiae, CIV is found solely in an SC with cytochrome bc1 (complex III, CIII). Here, we present the cryogenic electron microscopy (cryo-EM) structure of S. cerevisiae CIV in a III2IV2 SC at 3.3 Å resolution. While overall similarity to mammalian homologs is high, we found notable differences in the supernumerary subunits Cox26 and Cox13; the latter exhibits a unique arrangement that precludes CIV dimerization as seen in bovine. A conformational shift in the matrix domain of Cox5A—involved in allosteric inhibition by ATP—may arise from its association with CIII. The CIII–CIV arrangement highlights a conserved interaction interface of CIII, albeit one occupied by complex I in mammalian respirasomes. We discuss our findings in the context of the potential impact of SC formation on CIV regulation.	Dec 2018
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	A PxL motif promotes timely cell cycle substrate dephosphorylation by the Cdc14 phosphatase Meghna Kataria , Stephane Mouilleron , Moon-hyeong Seo , Carles Corbi-verge , Philip M. Kim , Frank Uhlmann Nature Structural & Molecular Biology 434 DOI: 10.1038/s41594-018-0152-3 Diamond Proposal Number(s): [13775] Show Abstract ▼ Abstract: The cell division cycle consists of a series of temporally ordered events. Cell cycle kinases and phosphatases provide key regulatory input, but how the correct substrate phosphorylation and dephosphorylation timing is achieved is incompletely understood. Here we identify a PxL substrate recognition motif that instructs dephosphorylation by the budding yeast Cdc14 phosphatase during mitotic exit. The PxL motif was prevalent in Cdc14-binding peptides enriched in a phage display screen of native disordered protein regions. PxL motif removal from the Cdc14 substrate Cbk1 delays its dephosphorylation, whereas addition of the motif advances dephosphorylation of otherwise late Cdc14 substrates. Crystal structures of Cdc14 bound to three PxL motif substrate peptides provide a molecular explanation for PxL motif recognition on the phosphatase surface. Our results illustrate the sophistication of phosphatase–substrate interactions and identify them as an important determinant of ordered cell cycle progression.	Nov 2018
B21-High Throughput SAXS I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis of meiotic chromosome synapsis through SYCP1 self-assembly James M. Dunce , Orla M. Dunne , Matthew Ratcliff , Claudia Millán , Suzanne Madgwick , Isabel Uson , Owen R. Davies Nature Structural & Molecular Biology 7 DOI: 10.1038/s41594-018-0078-9 Diamond Proposal Number(s): [9948, 13587, 14435, 15580, 15897] Open Access Show Abstract ▼ Abstract: Meiotic chromosomes adopt unique structures in which linear arrays of chromatin loops are bound together in homologous chromosome pairs by a supramolecular protein assembly, the synaptonemal complex. This three-dimensional scaffold provides the essential structural framework for genetic exchange by crossing over and subsequent homolog segregation. The core architecture of the synaptonemal complex is provided by SYCP1. Here we report the structure and self-assembly mechanism of human SYCP1 through X-ray crystallographic and biophysical studies. SYCP1 has an obligate tetrameric structure in which an N-terminal four-helical bundle bifurcates into two elongated C-terminal dimeric coiled-coils. This building block assembles into a zipper-like lattice through two self-assembly sites. N-terminal sites undergo cooperative head-to-head assembly in the midline, while C-terminal sites interact back to back on the chromosome axis. Our work reveals the underlying molecular structure of the synaptonemal complex in which SYCP1 self-assembly generates a supramolecular lattice that mediates meiotic chromosome synapsis.	Jun 2018

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