44 results on '"Benlekbir S"'
Search Results
2. Cleaved Ycf1p Monomer in the Beta Conformation
- Author
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Bickers, S.C., primary, Benlekbir, S., additional, Rubinstein, J.L., additional, and Kanelis, V., additional
- Published
- 2024
- Full Text
- View/download PDF
3. Cleaved Ycf1p Dimer in the IFwide-beta conformation
- Author
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Bickers, S.C., primary, Benlekbir, S., additional, Rubinstein, J.L., additional, and Kanelis, V., additional
- Published
- 2024
- Full Text
- View/download PDF
4. Cryo-EM structure of bafilomycin A1 bound to yeast VO V-ATPase
- Author
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Keon, K.A., primary, Rubinstein, J.L., additional, Benlekbir, S., additional, Kirsch, S.H., additional, and Muller, R., additional
- Published
- 2022
- Full Text
- View/download PDF
5. Cryo-EM structure of archazolid A bound to yeast VO V-ATPase
- Author
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Keon, K.A., primary, Rubinstein, J.L., additional, Benlekbir, S., additional, Kirsch, S.H., additional, and Muller, R., additional
- Published
- 2022
- Full Text
- View/download PDF
6. HAADF-TEM Tomography of the precipitation state in an Al-Zn-Mg alloy
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Epicier, T., Benlekbir, S., Danoix, F., Luysberg, Martina, editor, Tillmann, Karsten, editor, and Weirich, Thomas, editor
- Published
- 2008
- Full Text
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7. 3D-Geometrical and chemical quantification of Au@SiOx nano-composites in HAADF-STEM imaging mode
- Author
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Benlekbir, S., Epicier, T., Martini, M., Perriat, P., Luysberg, Martina, editor, Tillmann, Karsten, editor, and Weirich, Thomas, editor
- Published
- 2008
- Full Text
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8. Cryo-EM structure of the HCoV-229E spike glycoprotein
- Author
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Li, Z., primary, Benlekbir, S., additional, Rubinstein, J.L., additional, and Rini, J.M., additional
- Published
- 2019
- Full Text
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9. Structure of a functional obligate respiratory supercomplex from Mycobacterium smegmatis
- Author
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Wiseman, B., primary, Nitharwal, R.G., additional, Fedotovskaya, O., additional, Schafer, J., additional, Guo, H., additional, Kuang, Q., additional, Benlekbir, S., additional, Sjostrand, D., additional, Adelroth, P., additional, Rubinstein, J.L., additional, Brzezinski, P., additional, and Hogbom, M., additional
- Published
- 2018
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10. Structure of Alternative Complex III from Flavobacterium johnsoniae (Wild Type)
- Author
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Sun, C., primary, Benlekbir, S., additional, Venkatakrishnan, P., additional, Yuhang, W., additional, Tajkhorshid, E., additional, Rubinstein, J.L., additional, and Gennis, R.B., additional
- Published
- 2018
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11. Atomic model for the membrane-embedded motor of a eukaryotic V-ATPase
- Author
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Mazhab-Jafari, M.T., primary, Rohou, A., additional, Schmidt, C., additional, Bueler, S.A., additional, Benlekbir, S., additional, Robinson, C.V., additional, and Rubinstein, J.L., additional
- Published
- 2016
- Full Text
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12. Structural model of 53BP1 bound to a ubiquitylated and methylated nucleosome, at 4.5 A resolution
- Author
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Wilson, M.D., primary, Benlekbir, S., additional, Sicheri, F., additional, Rubinstein, J.L., additional, and Durocher, D., additional
- Published
- 2016
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13. Yeast V-ATPase state 2
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Zhao, J., primary, Benlekbir, S., additional, and Rubinstein, J.L., additional
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- 2015
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14. Yeast V-ATPase state 3
- Author
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Zhao, J., primary, Benlekbir, S., additional, and Rubinstein, J.L., additional
- Published
- 2015
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15. Yeast V-ATPase state 1
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Zhao, J., primary, Benlekbir, S., additional, and Rubinstein, J.L., additional
- Published
- 2015
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16. Synthèse de Catalyseurs Pd de Morphologie Contrôlée Supportés sur Al2O3 - Applications en Hydrogénation Sélective de Dioléfines et d'Aldéhydes alpha, béta-Insaturés
- Author
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Berhault, G., Bausach, M., Aouine, M., Benlekbir, S., Epicier, T., Institut de recherches sur la catalyse et l'environnement de Lyon (IRCELYON), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), 22 mai 2007, IRCELYON, ProductionsScientifiques, and 23 mai 2007
- Subjects
[CHIM.CATA] Chemical Sciences/Catalysis ,[SDE.ES] Environmental Sciences/Environmental and Society ,[CHIM.CATA]Chemical Sciences/Catalysis ,[SDE.ES]Environmental Sciences/Environmental and Society - Published
- 2007
17. 3D-Geometrical and chemical quantification of Au@SiOx nano-composites in HAADF-STEM imaging mode
- Author
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Benlekbir, S., primary, Epicier, T., additional, Martini, M., additional, and Perriat, P., additional
- Full Text
- View/download PDF
18. HAADF-TEM Tomography of the precipitation state in an Al-Zn-Mg alloy
- Author
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Epicier, T., primary, Benlekbir, S., additional, and Danoix, F., additional
- Full Text
- View/download PDF
19. STEM HAADF electron tomography of palladium nanoparticles with complex shapes
- Author
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Benlekbir, S., primary, Epicier, T., additional, Bausach, M., additional, Aouine, M., additional, and Berhault, G., additional
- Published
- 2009
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20. STEM HAADF electron tomography of palladium nanoparticles with complex shapes
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Benlekbir, S., Epicier, T., Bausach, M., Aouine, M., Berhault, G., Benlekbir, S., Epicier, T., Bausach, M., Aouine, M., and Berhault, G.
- Abstract
Palladium nanoparticles, with potential interest for various applications in catalysis, have been studied by Transmission Electron Microscopy (TEM). In order to ascertain the exact morphology of these particles, electron tomography was perfomed in the Scanning TEM, High Angle Annular Dark Field (STEM-HAADF) imaging mode. Several geometrical forms have thus been characterized three-dimensionally at the nanometre scale, among them pentagonal rods and more complex bipyramidal nanocrystals.
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21. Structure and dynamics of a pentameric KCTD5/CUL3/Gβγ E3 ubiquitin ligase complex.
- Author
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Nguyen DM, Rath DH, Devost D, Pétrin D, Rizk R, Ji AX, Narayanan N, Yong D, Zhai A, Kuntz DA, Mian MUQ, Pomroy NC, Keszei AFA, Benlekbir S, Mazhab-Jafari MT, Rubinstein JL, Hébert TE, and Privé GG
- Subjects
- Protein Binding, Ubiquitination, Ubiquitin metabolism, Cullin Proteins genetics, Cullin Proteins metabolism, Ubiquitin-Protein Ligases metabolism, Carrier Proteins metabolism
- Abstract
Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3
KCTD5 ) that ubiquitylates Gβγ and reduces Gβγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD /Gβ1 γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB /CUL3NTD and KCTD5CTD /Gβγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gβγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gβ and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD /Gβγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex., Competing Interests: Competing interests statement:The authors declare no competing interest.- Published
- 2024
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22. The Sec1-Munc18 protein VPS33B forms a uniquely bidirectional complex with VPS16B.
- Author
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Liu RJY, Al-Molieh Y, Chen SZ, Drobac M, Urban D, Chen CH, Yao HHY, Geng RSQ, Li L, Pluthero FG, Benlekbir S, Rubinstein JL, and Kahr WHA
- Subjects
- Humans, SNARE Proteins genetics, Syndrome, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, Munc18 Proteins, Renal Insufficiency
- Abstract
Loss-of-function variants of vacuolar protein sorting proteins VPS33B and VPS16B (VIPAS39) are causative for arthrogryposis, renal dysfunction, and cholestasis syndrome, where early lethality of patients indicates that VPS33B and VPS16B play essential cellular roles. VPS33B is a member of the Sec1-Munc18 protein family and thought to facilitate vesicular fusion via interaction with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, like its paralog VPS33A in the homotypic fusion and vacuole sorting complex. VPS33B and VPS16B are known to associate, but little is known about the composition, structure, or function of the VPS33B-VPS16B complex. We show here that human VPS33B-VPS16B is a high molecular weight complex, which we expressed in yeast to perform structural, composition, and stability analysis. Circular dichroism data indicate VPS33B-VPS16B has a well-folded α-helical secondary structure, and size-exclusion chromatography-multiangle light scattering revealed a molecular weight of ∼315 kDa. Quantitative immunoblotting indicated a VPS33B:VPS16B ratio of 2:3. Expression of arthrogryposis, renal dysfunction, and cholestasis syndrome-causing VPS33B missense variants showed L30P disrupts complex formation but not S243F or H344D. Truncated VPS16B (amino acids 143 to 316) was sufficient to form a complex with VPS33B. Small-angle X-ray scattering and negative-staining EM revealed a two-lobed shape for VPS33B-VPS16B. Avidin tagging indicated that each lobe contains a VPS33B molecule, and they are oriented in opposite directions. We propose a structure for VPS33B-VPS16B that allows the VPS33B at each end to interact with separate SNARE bundles and/or SNAREpins, plus associated membrane components. These observations reveal the only known potentially bidirectional Sec1-Munc18 protein complex., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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23. Cryo-EM of the Yeast V O Complex Reveals Distinct Binding Sites for Macrolide V-ATPase Inhibitors.
- Author
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Keon KA, Benlekbir S, Kirsch SH, Müller R, and Rubinstein JL
- Subjects
- Binding Sites, Cryoelectron Microscopy, Macrolides pharmacology, Protons, Saccharomyces cerevisiae metabolism, Vacuolar Proton-Translocating ATPases chemistry
- Abstract
Vacuolar-type adenosine triphosphatases (V-ATPases) are proton pumps found in almost all eukaryotic cells. These enzymes consist of a soluble catalytic V
1 region that hydrolyzes ATP and a membrane-embedded VO region responsible for proton translocation. V-ATPase activity leads to acidification of endosomes, phagosomes, lysosomes, secretory vesicles, and the trans-Golgi network, with extracellular acidification occurring in some specialized cells. Small-molecule inhibitors of V-ATPase have played a crucial role in elucidating numerous aspects of cell biology by blocking acidification of intracellular compartments, while therapeutic use of V-ATPase inhibitors has been proposed for the treatment of cancer, osteoporosis, and some infections. Here, we determine structures of the isolated VO complex from Saccharomyces cerevisiae bound to two well-known macrolide inhibitors: bafilomycin A1 and archazolid A. The structures reveal different binding sites for the inhibitors on the surface of the proton-carrying c ring, with only a small amount of overlap between the two sites. Binding of both inhibitors is mediated primarily through van der Waals interactions in shallow pockets and suggests that the inhibitors block rotation of the ring. Together, these structures indicate the existence of a large chemical space available for V-ATPase inhibitors that block acidification by binding the c ring.- Published
- 2022
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24. Multivalency transforms SARS-CoV-2 antibodies into ultrapotent neutralizers.
- Author
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Rujas E, Kucharska I, Tan YZ, Benlekbir S, Cui H, Zhao T, Wasney GA, Budylowski P, Guvenc F, Newton JC, Sicard T, Semesi A, Muthuraman K, Nouanesengsy A, Aschner CB, Prieto K, Bueler SA, Youssef S, Liao-Chan S, Glanville J, Christie-Holmes N, Mubareka S, Gray-Owen SD, Rubinstein JL, Treanor B, and Julien JP
- Subjects
- Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal genetics, Antibodies, Monoclonal immunology, Antibodies, Neutralizing chemistry, Antibodies, Viral immunology, Antibody Specificity, Apoferritins chemistry, Biological Availability, Epitope Mapping, Humans, Immunoglobulin G immunology, Male, Mice, Inbred BALB C, Mice, Inbred C57BL, Protein Engineering methods, Protein Subunits chemistry, Spike Glycoprotein, Coronavirus immunology, Tissue Distribution, Mice, Antibodies, Monoclonal pharmacology, Antibodies, Neutralizing immunology, Antibodies, Viral chemistry, SARS-CoV-2 immunology
- Abstract
SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC
50 ) values as low as 9 × 10- 14 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.- Published
- 2021
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25. Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory region of ABCC transporters.
- Author
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Bickers SC, Benlekbir S, Rubinstein JL, and Kanelis V
- Subjects
- ATP-Binding Cassette Transporters genetics, ATP-Binding Cassette Transporters metabolism, Binding Sites, Cell Membrane metabolism, Cloning, Molecular, Cryoelectron Microscopy, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Humans, Models, Molecular, Multidrug Resistance-Associated Protein 2 chemistry, Multidrug Resistance-Associated Protein 2 genetics, Multidrug Resistance-Associated Protein 2 metabolism, Multidrug Resistance-Associated Proteins genetics, Multidrug Resistance-Associated Proteins metabolism, Phosphorylation, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Sequence Homology, Amino Acid, Sulfonylurea Receptors chemistry, Sulfonylurea Receptors genetics, Sulfonylurea Receptors metabolism, ATP-Binding Cassette Transporters chemistry, Multidrug Resistance-Associated Proteins chemistry, Protein Processing, Post-Translational, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins chemistry
- Abstract
ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K
+ channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a ∼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease., Competing Interests: The authors declare no competing interest.- Published
- 2021
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26. Structural basis of substrate recognition and thermal protection by a small heat shock protein.
- Author
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Yu C, Leung SKP, Zhang W, Lai LTF, Chan YK, Wong MC, Benlekbir S, Cui Y, Jiang L, and Lau WCY
- Subjects
- Arabidopsis genetics, Arabidopsis Proteins metabolism, Cryoelectron Microscopy, Heat-Shock Proteins chemistry, Heat-Shock Proteins metabolism, Heat-Shock Proteins, Small genetics, Heat-Shock Response, Models, Molecular, Protein Folding, Transferases chemistry, Transferases metabolism, Arabidopsis metabolism, Heat-Shock Proteins, Small chemistry, Heat-Shock Proteins, Small metabolism
- Abstract
Small heat shock proteins (sHsps) bind unfolding proteins, thereby playing a pivotal role in the maintenance of proteostasis in virtually all living organisms. Structural elucidation of sHsp-substrate complexes has been hampered by the transient and heterogeneous nature of their interactions, and the precise mechanisms underlying substrate recognition, promiscuity, and chaperone activity of sHsps remain unclear. Here we show the formation of a stable complex between Arabidopsis thaliana plastid sHsp, Hsp21, and its natural substrate 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) under heat stress, and report cryo-electron microscopy structures of Hsp21, DXPS and Hsp21-DXPS complex at near-atomic resolution. Monomeric Hsp21 binds across the dimer interface of DXPS and engages in multivalent interactions by recognizing highly dynamic structural elements in DXPS. Hsp21 partly unfolds its central α-crystallin domain to facilitate binding of DXPS, which preserves a native-like structure. This mode of interaction suggests a mechanism of sHsps anti-aggregation activity towards a broad range of substrates.
- Published
- 2021
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27. Revised subunit order of mammalian septin complexes explains their in vitro polymerization properties.
- Author
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Soroor F, Kim MS, Palander O, Balachandran Y, Collins RF, Benlekbir S, Rubinstein JL, and Trimble WS
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- Animals, Cell Cycle Proteins metabolism, Cytoskeleton metabolism, HeLa Cells, Humans, Mammals metabolism, Polymerization, Septins metabolism, Septins physiology
- Abstract
Septins are conserved GTP-binding cytoskeletal proteins that polymerize into filaments by end-to-end joining of hetero-oligomeric complexes. In human cells, both hexamers and octamers exist, and crystallography studies predicted the order of the hexamers to be SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7, while octamers are thought to have the same core, but with SEPT9 at the ends. However, based on this septin organization, octamers and hexamers would not be expected to copolymerize due to incompatible ends. Here we isolated hexamers and octamers of specific composition from human cells and show that hexamers and octamers polymerize individually and, surprisingly, with each other. Binding of the Borg homology domain 3 (BD3) domain of Borg3 results in distinctive clustering of each filament type. Moreover, we show that the organization of hexameric and octameric complexes is inverted compared with its original prediction. This revised septin organization is congruent with the organization and behavior of yeast septins suggesting that their properties are more conserved than was previously thought.
- Published
- 2021
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28. Electron-event representation data enable efficient cryoEM file storage with full preservation of spatial and temporal resolution.
- Author
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Guo H, Franken E, Deng Y, Benlekbir S, Singla Lezcano G, Janssen B, Yu L, Ripstein ZA, Tan YZ, and Rubinstein JL
- Abstract
Direct detector device (DDD) cameras have revolutionized electron cryomicroscopy (cryoEM) with their high detective quantum efficiency (DQE) and output of movie data. A high ratio of camera frame rate (frames per second) to camera exposure rate (electrons per pixel per second) allows electron counting, which further improves the DQE and enables the recording of super-resolution information. Movie output also allows the correction of specimen movement and compensation for radiation damage. However, these movies come at the cost of producing large volumes of data. It is common practice to sum groups of successive camera frames to reduce the final frame rate, and therefore the file size, to one suitable for storage and image processing. This reduction in the temporal resolution of the camera requires decisions to be made during data acquisition that may result in the loss of information that could have been advantageous during image analysis. Here, experimental analysis of a new electron-event representation (EER) data format for electron-counting DDD movies is presented, which is enabled by new hardware developed by Thermo Fisher Scientific for their Falcon DDD cameras. This format enables the recording of DDD movies at the raw camera frame rate without sacrificing either spatial or temporal resolution. Experimental data demonstrate that the method retains super-resolution information and allows the correction of specimen movement at the physical frame rate of the camera while maintaining manageable file sizes. The EER format will enable the development of new methods that can utilize the full spatial and temporal resolution of DDD cameras., (© Hui Guo et al. 2020.)
- Published
- 2020
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29. Subnanometer resolution cryo-EM structure of Arabidopsis thaliana ATG9.
- Author
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Lai LTF, Yu C, Wong JSK, Lo HS, Benlekbir S, Jiang L, and Lau WCY
- Subjects
- Arabidopsis Proteins ultrastructure, Autophagy-Related Proteins ultrastructure, Membrane Proteins ultrastructure, Models, Molecular, Protein Multimerization, Protein Structure, Secondary, Structural Homology, Protein, Arabidopsis metabolism, Arabidopsis ultrastructure, Arabidopsis Proteins metabolism, Autophagy-Related Proteins metabolism, Cryoelectron Microscopy, Membrane Proteins metabolism, Nanotechnology
- Abstract
Macroautophagy/autophagy is an essential process for the maintenance of cellular homeostasis by recycling macromolecules under normal and stress conditions. ATG9 (autophagy related 9) is the only integral membrane protein in the autophagy core machinery and has a central role in mediating autophagosome formation. In cells, ATG9 exists on mobile vesicles that traffic to the growing phagophore, providing an essential membrane source for the formation of autophagosomes. Here we report the three-dimensional structure of ATG9 from Arabidopsis thaliana at 7.8 Å resolution, determined by single particle cryo-electron microscopy. ATG9 organizes into a homotrimer, with each protomer contributing at least six transmembrane α-helices. At the center of the trimer, the protomers interact via their membrane-embedded and C-terminal cytoplasmic regions. Combined with prediction of protein contacts using sequence co-evolutionary information, the structure provides molecular insights into the ATG9 architecture and testable hypotheses for the molecular mechanism of autophagy progression regulated by ATG9. Abbreviations: 2D: 2-dimensional; 3D: 3-dimensional; AtATG9: Arabidopsis ATG9; Atg: autophagy-related; ATG9: autophagy-related protein 9; cryo-EM: cryo-electron microscopy; DDM: dodecyl maltoside; GraDeR: gradient-based detergent removal; LMNG: lauryl maltose-neopentyl glycol; PAS: phagophore assembly site; PtdIns3K: phosphatidylinositol 3-kinase.
- Published
- 2020
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30. Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device.
- Author
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Rubinstein JL, Guo H, Ripstein ZA, Haydaroglu A, Au A, Yip CM, Di Trani JM, Benlekbir S, and Kwok T
- Subjects
- Software, Vitrification, Cryoelectron Microscopy instrumentation, Macromolecular Substances chemistry, Proteins chemistry, Specimen Handling instrumentation
- Abstract
Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments., (open access.)
- Published
- 2019
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- View/download PDF
31. Multiple conformations facilitate PilT function in the type IV pilus.
- Author
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McCallum M, Benlekbir S, Nguyen S, Tammam S, Rubinstein JL, Burrows LL, and Howell PL
- Subjects
- Adenosine Triphosphatases chemistry, Adenosine Triphosphatases genetics, Bacterial Proteins chemistry, Bacterial Proteins genetics, Caulobacter chemistry, Caulobacter genetics, Crystallography, X-Ray, Fimbriae, Bacterial genetics, Multigene Family, Protein Conformation, Adenosine Triphosphatases metabolism, Bacterial Proteins metabolism, Caulobacter metabolism, Fimbriae, Bacterial metabolism
- Abstract
Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C
2 symmetry; however, most of these ATPases crystallize with either C3 or C6 symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C2, C3 , and C6 conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.- Published
- 2019
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32. The human coronavirus HCoV-229E S-protein structure and receptor binding.
- Author
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Li Z, Tomlinson AC, Wong AH, Zhou D, Desforges M, Talbot PJ, Benlekbir S, Rubinstein JL, and Rini JM
- Subjects
- Cryoelectron Microscopy, Crystallography, X-Ray, Humans, Protein Binding, Protein Domains, CD13 Antigens chemistry, CD13 Antigens metabolism, Coronavirus 229E, Human enzymology, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus metabolism
- Abstract
The coronavirus S-protein mediates receptor binding and fusion of the viral and host cell membranes. In HCoV-229E, its receptor binding domain (RBD) shows extensive sequence variation but how S-protein function is maintained is not understood. Reported are the X-ray crystal structures of Class III-V RBDs in complex with human aminopeptidase N (hAPN), as well as the electron cryomicroscopy structure of the 229E S-protein. The structures show that common core interactions define the specificity for hAPN and that the peripheral RBD sequence variation is accommodated by loop plasticity. The results provide insight into immune evasion and the cross-species transmission of 229E and related coronaviruses. We also find that the 229E S-protein can expose a portion of its helical core to solvent. This is undoubtedly facilitated by hydrophilic subunit interfaces that we show are conserved among coronaviruses. These interfaces likely play a role in the S-protein conformational changes associated with membrane fusion., Competing Interests: ZL, AT, AW, DZ, MD, PT, SB, JR, JR No competing interests declared, (© 2019, Li et al.)
- Published
- 2019
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33. Structure of a functional obligate complex III 2 IV 2 respiratory supercomplex from Mycobacterium smegmatis.
- Author
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Wiseman B, Nitharwal RG, Fedotovskaya O, Schäfer J, Guo H, Kuang Q, Benlekbir S, Sjöstrand D, Ädelroth P, Rubinstein JL, Brzezinski P, and Högbom M
- Subjects
- Cryoelectron Microscopy, Electron Transport, Electron Transport Chain Complex Proteins chemistry, Electron Transport Chain Complex Proteins metabolism, Electron Transport Complex III chemistry, Mycobacterium smegmatis cytology, Oxidation-Reduction, Oxygen, Protein Structure, Tertiary, Cell Respiration physiology, Electron Transport Chain Complex Proteins physiology, Electron Transport Complex III physiology, Models, Molecular, Mycobacterium smegmatis metabolism
- Abstract
In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III
2 IV2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Qo and Qi sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c1 and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.- Published
- 2018
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34. Structure of the alternative complex III in a supercomplex with cytochrome oxidase.
- Author
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Sun C, Benlekbir S, Venkatakrishnan P, Wang Y, Hong S, Hosler J, Tajkhorshid E, Rubinstein JL, and Gennis RB
- Subjects
- Cysteine chemistry, Cysteine metabolism, Cytochrome c Group metabolism, Cytochromes a metabolism, Cytochromes a3 metabolism, Electron Transport Complex III metabolism, Heme analogs & derivatives, Heme chemistry, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Lipids chemistry, Models, Molecular, Nanostructures chemistry, Nanostructures ultrastructure, Oxidation-Reduction, Protein Subunits chemistry, Protein Subunits metabolism, Cryoelectron Microscopy, Cytochrome c Group chemistry, Cytochrome c Group ultrastructure, Cytochromes a chemistry, Cytochromes a ultrastructure, Cytochromes a3 chemistry, Cytochromes a3 ultrastructure, Electron Transport Complex III chemistry, Electron Transport Complex III ultrastructure, Flavobacterium enzymology
- Abstract
Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria
1-3 . Like complex III (also known as the bc1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity4-7 . Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily8 , including the proton pump polysulfide reductase9,10 . We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer11-14 , both as an independent enzyme and as a functional 1:1 supercomplex with an aa3 -type cytochrome c oxidase (cyt aa3 ). We determined the structure of ACIII to 3.4 Å resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe-4S] cluster, a [4Fe-4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer.- Published
- 2018
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35. WITHDRAWN: Characterization of the supercomplex formed by the alternative complex III and the terminal aa 3 oxidase from Flavobacterium johnsoniae isolated in styrene:maleic acid copolymer nanodiscs.
- Author
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Venkatakrishnan P, Benlekbir S, Sun C, Hong S, Hosler J, Rubinstein J, and Gennis RB
- Abstract
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal., (Copyright © 2018.)
- Published
- 2018
- Full Text
- View/download PDF
36. Molecular basis of human CD22 function and therapeutic targeting.
- Author
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Ereño-Orbea J, Sicard T, Cui H, Mazhab-Jafari MT, Benlekbir S, Guarné A, Rubinstein JL, and Julien JP
- Subjects
- Antibodies, Monoclonal, Humanized ultrastructure, Crystallography, X-Ray, Humans, Lectins immunology, Microscopy, Electron, Molecular Targeted Therapy, Protein Conformation, Sialic Acid Binding Ig-like Lectin 2 ultrastructure, Autoimmunity immunology, B-Lymphocytes immunology, Immunity, Humoral immunology, Sialic Acid Binding Ig-like Lectin 2 immunology
- Abstract
CD22 maintains a baseline level of B-cell inhibition to keep humoral immunity in check. As a B-cell-restricted antigen, CD22 is targeted in therapies against dysregulated B cells that cause autoimmune diseases and blood cancers. Here we report the crystal structure of human CD22 at 2.1 Å resolution, which reveals that specificity for α2-6 sialic acid ligands is dictated by a pre-formed β-hairpin as a unique mode of recognition across sialic acid-binding immunoglobulin-type lectins. The CD22 ectodomain adopts an extended conformation that facilitates concomitant CD22 nanocluster formation on B cells and binding to trans ligands to avert autoimmunity in mammals. We structurally delineate the CD22 site targeted by the therapeutic antibody epratuzumab at 3.1 Å resolution and determine a critical role for CD22 N-linked glycosylation in antibody engagement. Our studies provide molecular insights into mechanisms governing B-cell inhibition and valuable clues for the design of immune modulators in B-cell dysfunction.The B-cell-specific co-receptor CD22 is a therapeutic target for depleting dysregulated B cells. Here the authors structurally characterize the ectodomain of CD22 and present its crystal structure with the bound therapeutic antibody epratuzumab, which gives insights into the mechanism of inhibition of B-cell activation.
- Published
- 2017
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37. Atomic model for the membrane-embedded V O motor of a eukaryotic V-ATPase.
- Author
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Mazhab-Jafari MT, Rohou A, Schmidt C, Bueler SA, Benlekbir S, Robinson CV, and Rubinstein JL
- Subjects
- Adenosine Triphosphate chemistry, Adenosine Triphosphate metabolism, Arginine chemistry, Arginine metabolism, Glutamic Acid chemistry, Glutamic Acid metabolism, Hydrolysis, Models, Molecular, Protein Subunits chemistry, Protein Subunits metabolism, Protons, Rotation, Saccharomyces cerevisiae chemistry, Cryoelectron Microscopy, Saccharomyces cerevisiae ultrastructure, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins ultrastructure, Vacuolar Proton-Translocating ATPases chemistry, Vacuolar Proton-Translocating ATPases ultrastructure
- Abstract
Vacuolar-type ATPases (V-ATPases) are ATP-powered proton pumps involved in processes such as endocytosis, lysosomal degradation, secondary transport, TOR signalling, and osteoclast and kidney function. ATP hydrolysis in the soluble catalytic V
1 region drives proton translocation through the membrane-embedded VO region via rotation of a rotor subcomplex. Variability in the structure of the intact enzyme has prevented construction of an atomic model for the membrane-embedded motor of any rotary ATPase. We induced dissociation and auto-inhibition of the V1 and VO regions of the V-ATPase by starving the yeast Saccharomyces cerevisiae, allowing us to obtain a ~3.9-Å resolution electron cryomicroscopy map of the VO complex and build atomic models for the majority of its subunits. The analysis reveals the structures of subunits ac8 c'c″de and a protein that we identify and propose to be a new subunit (subunit f). A large cavity between subunit a and the c-ring creates a cytoplasmic half-channel for protons. The c-ring has an asymmetric distribution of proton-carrying Glu residues, with the Glu residue of subunit c″ interacting with Arg735 of subunit a. The structure suggests sequential protonation and deprotonation of the c-ring, with ATP-hydrolysis-driven rotation causing protonation of a Glu residue at the cytoplasmic half-channel and subsequent deprotonation of a Glu residue at a luminal half-channel.- Published
- 2016
- Full Text
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38. YphC and YsxC GTPases assist the maturation of the central protuberance, GTPase associated region and functional core of the 50S ribosomal subunit.
- Author
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Ni X, Davis JH, Jain N, Razi A, Benlekbir S, McArthur AG, Rubinstein JL, Britton RA, Williamson JR, and Ortega J
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins ultrastructure, Cryoelectron Microscopy, GTP Phosphohydrolases chemistry, GTP Phosphohydrolases ultrastructure, Kinetics, Mass Spectrometry, Protein Conformation, Protein Structure, Secondary, Protein Subunits metabolism, Ribosomal Proteins chemistry, Ribosomal Proteins ultrastructure, Ribosome Subunits, Large, Bacterial ultrastructure, Bacterial Proteins metabolism, GTP Phosphohydrolases metabolism, Ribosomal Proteins metabolism, Ribosome Subunits, Large, Bacterial metabolism
- Abstract
YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45SYphC and 44.5SYsxC particles. Quantitative mass spectrometry analysis and the 5-6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2016
- Full Text
- View/download PDF
39. The structural basis of modified nucleosome recognition by 53BP1.
- Author
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Wilson MD, Benlekbir S, Fradet-Turcotte A, Sherker A, Julien JP, McEwan A, Noordermeer SM, Sicheri F, Rubinstein JL, and Durocher D
- Subjects
- DNA Breaks, Double-Stranded, DNA Repair, Humans, Methylation, Models, Molecular, Nucleosomes chemistry, Nucleosomes genetics, Pliability, Protein Multimerization, Protein Structure, Tertiary, Substrate Specificity, Tumor Suppressor p53-Binding Protein 1, Ubiquitin metabolism, Ubiquitination, Cryoelectron Microscopy, Histones chemistry, Histones metabolism, Intracellular Signaling Peptides and Proteins chemistry, Intracellular Signaling Peptides and Proteins metabolism, Nucleosomes metabolism, Nucleosomes ultrastructure
- Abstract
DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.
- Published
- 2016
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40. Structural Insights into KCTD Protein Assembly and Cullin3 Recognition.
- Author
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Ji AX, Chu A, Nielsen TK, Benlekbir S, Rubinstein JL, and Privé GG
- Subjects
- Co-Repressor Proteins, Cryoelectron Microscopy, Crystallography, X-Ray, Models, Molecular, Protein Binding, Protein Conformation, Protein Multimerization, Cullin Proteins metabolism, Macromolecular Substances chemistry, Macromolecular Substances metabolism, Potassium Channels chemistry, Potassium Channels metabolism, Repressor Proteins chemistry, Repressor Proteins metabolism
- Abstract
Cullin3 (Cul3)-based ubiquitin E3 ligase complexes catalyze the transfer of ubiquitin from an E2 enzyme to target substrate proteins. In these assemblies, the C-terminal region of Cul3 binds Rbx1/E2-ubiquitin, while the N-terminal region interacts with various BTB (bric-à-brac, tramtrack, broad complex) domain proteins that serve as substrate adaptors. Previous crystal structures of the homodimeric BTB proteins KLHL3, KLHL11 and SPOP in complex with the N-terminal domain of Cul3 revealed the features required for Cul3 recognition in these proteins. A second class of BTB-domain-containing proteins, the KCTD proteins, is also Cul3 substrate adaptors, but these do not share many of the previously identified determinants for Cul3 binding. We report the pentameric crystal structures of the KCTD1 and KCTD9 BTB domains and identify plasticity in the KCTD1 rings. We find that the KCTD proteins 5, 6, 9 and 17 bind to Cul3 with high affinity, while the KCTD proteins 1 and 16 do not have detectable binding. Finally, we confirm the 5:5 assembly of KCTD9/Cul3 complexes by cryo-electron microscopy and provide a molecular rationale for BTB-mediated Cul3 binding specificity in the KCTD family., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
41. Description and comparison of algorithms for correcting anisotropic magnification in cryo-EM images.
- Author
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Zhao J, Brubaker MA, Benlekbir S, and Rubinstein JL
- Subjects
- Algorithms, Cryoelectron Microscopy instrumentation, Imaging, Three-Dimensional methods, Multiprotein Complexes ultrastructure, Proteins ultrastructure, Thallium analysis, Anisotropy, Cryoelectron Microscopy methods, Image Enhancement methods, Multiprotein Complexes analysis, Proteins analysis
- Abstract
Single particle electron cryomicroscopy (cryo-EM) allows for structures of proteins and protein complexes to be determined from images of non-crystalline specimens. Cryo-EM data analysis requires electron microscope images of randomly oriented ice-embedded protein particles to be rotated and translated to allow for coherent averaging when calculating three-dimensional (3D) structures. Rotation of 2D images is usually done with the assumption that the magnification of the electron microscope is the same in all directions. However, due to electron optical aberrations, this condition is not met with some electron microscopes when used with the settings necessary for cryo-EM with a direct detector device (DDD) camera. Correction of images by linear interpolation in real space has allowed high-resolution structures to be calculated from cryo-EM images for symmetric particles. Here we describe and compare a simple real space method, a simple Fourier space method, and a somewhat more sophisticated Fourier space method to correct images for a measured anisotropy in magnification. Further, anisotropic magnification causes contrast transfer function (CTF) parameters estimated from image power spectra to have an apparent systematic astigmatism. To address this problem we develop an approach to adjust CTF parameters measured from distorted images so that they can be used with corrected images. The effect of anisotropic magnification on CTF parameters provides a simple way of detecting magnification anisotropy in cryo-EM datasets., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
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42. Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase.
- Author
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Zhao J, Benlekbir S, and Rubinstein JL
- Subjects
- Adenosine Triphosphate metabolism, Biocatalysis, Cell Membrane chemistry, Cell Membrane enzymology, Cell Membrane metabolism, Lipid Bilayers metabolism, Models, Molecular, Pliability, Protein Conformation, Protein Subunits chemistry, Protein Subunits metabolism, Protons, Solubility, Vacuolar Proton-Translocating ATPases metabolism, Cryoelectron Microscopy, Rotation, Saccharomyces cerevisiae enzymology, Vacuolar Proton-Translocating ATPases chemistry, Vacuolar Proton-Translocating ATPases ultrastructure
- Abstract
Eukaryotic vacuolar H(+)-ATPases (V-ATPases) are rotary enzymes that use energy from hydrolysis of ATP to ADP to pump protons across membranes and control the pH of many intracellular compartments. ATP hydrolysis in the soluble catalytic region of the enzyme is coupled to proton translocation through the membrane-bound region by rotation of a central rotor subcomplex, with peripheral stalks preventing the entire membrane-bound region from turning with the rotor. The eukaryotic V-ATPase is the most complex rotary ATPase: it has three peripheral stalks, a hetero-oligomeric proton-conducting proteolipid ring, several subunits not found in other rotary ATPases, and is regulated by reversible dissociation of its catalytic and proton-conducting regions. Studies of ATP synthases, V-ATPases, and bacterial/archaeal V/A-ATPases have suggested that flexibility is necessary for the catalytic mechanism of rotary ATPases, but the structures of different rotational states have never been observed experimentally. Here we use electron cryomicroscopy to obtain structures for three rotational states of the V-ATPase from the yeast Saccharomyces cerevisiae. The resulting series of structures shows ten proteolipid subunits in the c-ring, setting the ATP:H(+) ratio for proton pumping by the V-ATPase at 3:10, and reveals long and highly tilted transmembrane α-helices in the a-subunit that interact with the c-ring. The three different maps reveal the conformational changes that occur to couple rotation in the symmetry-mismatched soluble catalytic region to the membrane-bound proton-translocating region. Almost all of the subunits of the enzyme undergo conformational changes during the transitions between these three rotational states. The structures of these states provide direct evidence that deformation during rotation enables the smooth transmission of power through rotary ATPases.
- Published
- 2015
- Full Text
- View/download PDF
43. Fabrication of carbon films with ∼ 500nm holes for cryo-EM with a direct detector device.
- Author
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Marr CR, Benlekbir S, and Rubinstein JL
- Subjects
- Carbon chemistry, Cryoelectron Microscopy instrumentation, Cryoelectron Microscopy methods, Image Processing, Computer-Assisted instrumentation, Image Processing, Computer-Assisted supply & distribution
- Abstract
Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500nm holes spaced 4μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
44. Structure of the vacuolar-type ATPase from Saccharomyces cerevisiae at 11-Å resolution.
- Author
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Benlekbir S, Bueler SA, and Rubinstein JL
- Subjects
- Cryoelectron Microscopy, Protein Conformation, Vacuolar Proton-Translocating ATPases metabolism, Vacuolar Proton-Translocating ATPases ultrastructure, Saccharomyces cerevisiae enzymology, Vacuolar Proton-Translocating ATPases chemistry
- Abstract
Vacuolar-type ATPases (V-type ATPases) in eukaryotic cells are large membrane protein complexes that acidify various intracellular compartments. The enzymes are regulated by dissociation of the V(1) and V(O) regions of the complex. Here we present the structure of the Saccharomyces cerevisiae V-type ATPase at 11-Å resolution by cryo-EM of protein particles in ice. The structure explains many cross-linking and protein interaction studies. Docking of crystal structures suggests that inhibition of ATPase activity by the dissociated V(1) region involves rearrangement of the N- and C-terminal domains of subunit H and also suggests how this inhibition is triggered upon dissociation. We provide support for this model by demonstrating that mutation of subunit H to increase the rigidity of the linker between its two domains decreases its ability to inhibit ATPase activity.
- Published
- 2012
- Full Text
- View/download PDF
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