21 results on '"Brilot AF"'
Search Results
2. Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps.
- Author
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Remesh SG, Merz GE, Brilot AF, Chio US, Rizo AN, Pospiech TH Jr, Lui I, Laurie MT, Glasgow J, Le CQ, Zhang Y, Diwanji D, Hernandez E, Lopez J, Mehmood H, Pawar KI, Pourmal S, Smith AM, Zhou F, DeRisi J, Kortemme T, Rosenberg OS, Glasgow A, Leung KK, Wells JA, and Verba KA
- Subjects
- Humans, SARS-CoV-2, Antibodies, Monoclonal, Protein Binding, Antibodies, Neutralizing, Angiotensin-Converting Enzyme 2, COVID-19
- Abstract
The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor-binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with stabilized Spike ectodomain. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high-affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high-affinity (0.53-4.2 nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron and Delta pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)
- Published
- 2023
- Full Text
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3. The ϕPA3 phage nucleus is enclosed by a self-assembling 2D crystalline lattice.
- Author
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Nieweglowska ES, Brilot AF, Méndez-Moran M, Kokontis C, Baek M, Li J, Cheng Y, Baker D, Bondy-Denomy J, and Agard DA
- Subjects
- Viral Proteins metabolism, CRISPR-Cas Systems, Bacteriophages genetics
- Abstract
To protect themselves from host attack, numerous jumbo bacteriophages establish a phage nucleus-a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the phage nucleus while other phage and host proteins are excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that nucleus fragments isolated from ϕPA3 infected Pseudomonas aeruginosa form a 2-dimensional lattice, having p2 or p4 symmetry. We further demonstrate that recombinantly purified primary Phage Nuclear Enclosure (PhuN) protein spontaneously assembles into similar 2D sheets with p2 and p4 symmetry. We resolve the dominant p2 symmetric state to 3.9 Å by cryo-EM. Our structure reveals a two-domain core, organized into quasi-symmetric tetramers. Flexible loops and termini mediate adaptable inter-tetramer contacts that drive subunit assembly into a lattice and enable the adoption of different symmetric states. While the interfaces between subunits are mostly well packed, two are open, forming channels that likely have functional implications for the transport of proteins, mRNA, and small molecules., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
4. Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps.
- Author
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Remesh SG, Merz GE, Brilot AF, Chio US, Rizo AN, Pospiech TH Jr, Lui I, Laurie MT, Glasgow J, Le CQ, Zhang Y, Diwanji D, Hernandez E, Lopez J, Pawar KI, Pourmal S, Smith AM, Zhou F, DeRisi J, Kortemme T, Rosenberg OS, Glasgow A, Leung KK, Wells JA, and Verba KA
- Abstract
The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta-pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.
- Published
- 2022
- Full Text
- View/download PDF
5. CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.
- Author
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Gupta M, Azumaya CM, Moritz M, Pourmal S, Diallo A, Merz GE, Jang G, Bouhaddou M, Fossati A, Brilot AF, Diwanji D, Hernandez E, Herrera N, Kratochvil HT, Lam VL, Li F, Li Y, Nguyen HC, Nowotny C, Owens TW, Peters JK, Rizo AN, Schulze-Gahmen U, Smith AM, Young ID, Yu Z, Asarnow D, Billesbølle C, Campbell MG, Chen J, Chen KH, Chio US, Dickinson MS, Doan L, Jin M, Kim K, Li J, Li YL, Linossi E, Liu Y, Lo M, Lopez J, Lopez KE, Mancino A, Moss FR 3rd, Paul MD, Pawar KI, Pelin A, Pospiech TH Jr, Puchades C, Remesh SG, Safari M, Schaefer K, Sun M, Tabios MC, Thwin AC, Titus EW, Trenker R, Tse E, Tsui TKM, Wang F, Zhang K, Zhang Y, Zhao J, Zhou F, Zhou Y, Zuliani-Alvarez L, Agard DA, Cheng Y, Fraser JS, Jura N, Kortemme T, Manglik A, Southworth DR, Stroud RM, Swaney DL, Krogan NJ, Frost A, Rosenberg OS, and Verba KA
- Abstract
The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.
- Published
- 2021
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6. CM1-driven assembly and activation of yeast γ-tubulin small complex underlies microtubule nucleation.
- Author
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Brilot AF, Lyon AS, Zelter A, Viswanath S, Maxwell A, MacCoss MJ, Muller EG, Sali A, Davis TN, and Agard DA
- Subjects
- Binding Sites, Calmodulin-Binding Proteins genetics, Calmodulin-Binding Proteins metabolism, Cryoelectron Microscopy methods, Crystallography, X-Ray methods, Cytoskeletal Proteins genetics, Cytoskeletal Proteins metabolism, Humans, Mass Spectrometry methods, Microtubule-Organizing Center, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Tubulin classification, Tubulin metabolism, Antigens, Nuclear genetics, Microtubules physiology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Tubulin chemistry, Tubulin genetics
- Abstract
Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In Saccharomyces cerevisiae , γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub-assemblies, which associate helically to template MT growth. γTuRC assembly provides a key point of regulation for the MT cytoskeleton. Here, we combine crosslinking mass spectrometry, X-ray crystallography, and cryo-EM structures of both monomeric and dimeric γTuSCs, and open and closed helical γTuRC assemblies in complex with Spc110p to elucidate the mechanisms of γTuRC assembly. γTuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent γTuSCs. By providing the highest resolution and most complete views of any γTuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human γTuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan γTuRC., Competing Interests: AB, AL, AZ, SV, AM, MM, EM, AS, TD, DA No competing interests declared, (© 2021, Brilot et al.)
- Published
- 2021
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7. An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.
- Author
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Schoof M, Faust B, Saunders RA, Sangwan S, Rezelj V, Hoppe N, Boone M, Billesbølle CB, Puchades C, Azumaya CM, Kratochvil HT, Zimanyi M, Deshpande I, Liang J, Dickinson S, Nguyen HC, Chio CM, Merz GE, Thompson MC, Diwanji D, Schaefer K, Anand AA, Dobzinski N, Zha BS, Simoneau CR, Leon K, White KM, Chio US, Gupta M, Jin M, Li F, Liu Y, Zhang K, Bulkley D, Sun M, Smith AM, Rizo AN, Moss F, Brilot AF, Pourmal S, Trenker R, Pospiech T, Gupta S, Barsi-Rhyne B, Belyy V, Barile-Hill AW, Nock S, Liu Y, Krogan NJ, Ralston CY, Swaney DL, García-Sastre A, Ott M, Vignuzzi M, Walter P, and Manglik A
- Subjects
- Angiotensin-Converting Enzyme 2 chemistry, Angiotensin-Converting Enzyme 2 immunology, Animals, Antibodies, Neutralizing chemistry, Antibodies, Viral chemistry, Antibody Affinity, Chlorocebus aethiops, Cryoelectron Microscopy, Humans, Neutralization Tests, Protein Binding, Protein Stability, Single-Domain Antibodies chemistry, Spike Glycoprotein, Coronavirus chemistry, Vero Cells, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Single-Domain Antibodies immunology, Spike Glycoprotein, Coronavirus immunology
- Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2020
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8. Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.
- Author
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Gordon DE, Hiatt J, Bouhaddou M, Rezelj VV, Ulferts S, Braberg H, Jureka AS, Obernier K, Guo JZ, Batra J, Kaake RM, Weckstein AR, Owens TW, Gupta M, Pourmal S, Titus EW, Cakir M, Soucheray M, McGregor M, Cakir Z, Jang G, O'Meara MJ, Tummino TA, Zhang Z, Foussard H, Rojc A, Zhou Y, Kuchenov D, Hüttenhain R, Xu J, Eckhardt M, Swaney DL, Fabius JM, Ummadi M, Tutuncuoglu B, Rathore U, Modak M, Haas P, Haas KM, Naing ZZC, Pulido EH, Shi Y, Barrio-Hernandez I, Memon D, Petsalaki E, Dunham A, Marrero MC, Burke D, Koh C, Vallet T, Silvas JA, Azumaya CM, Billesbølle C, Brilot AF, Campbell MG, Diallo A, Dickinson MS, Diwanji D, Herrera N, Hoppe N, Kratochvil HT, Liu Y, Merz GE, Moritz M, Nguyen HC, Nowotny C, Puchades C, Rizo AN, Schulze-Gahmen U, Smith AM, Sun M, Young ID, Zhao J, Asarnow D, Biel J, Bowen A, Braxton JR, Chen J, Chio CM, Chio US, Deshpande I, Doan L, Faust B, Flores S, Jin M, Kim K, Lam VL, Li F, Li J, Li YL, Li Y, Liu X, Lo M, Lopez KE, Melo AA, Moss FR 3rd, Nguyen P, Paulino J, Pawar KI, Peters JK, Pospiech TH Jr, Safari M, Sangwan S, Schaefer K, Thomas PV, Thwin AC, Trenker R, Tse E, Tsui TKM, Wang F, Whitis N, Yu Z, Zhang K, Zhang Y, Zhou F, Saltzberg D, Hodder AJ, Shun-Shion AS, Williams DM, White KM, Rosales R, Kehrer T, Miorin L, Moreno E, Patel AH, Rihn S, Khalid MM, Vallejo-Gracia A, Fozouni P, Simoneau CR, Roth TL, Wu D, Karim MA, Ghoussaini M, Dunham I, Berardi F, Weigang S, Chazal M, Park J, Logue J, McGrath M, Weston S, Haupt R, Hastie CJ, Elliott M, Brown F, Burness KA, Reid E, Dorward M, Johnson C, Wilkinson SG, Geyer A, Giesel DM, Baillie C, Raggett S, Leech H, Toth R, Goodman N, Keough KC, Lind AL, Klesh RJ, Hemphill KR, Carlson-Stevermer J, Oki J, Holden K, Maures T, Pollard KS, Sali A, Agard DA, Cheng Y, Fraser JS, Frost A, Jura N, Kortemme T, Manglik A, Southworth DR, Stroud RM, Alessi DR, Davies P, Frieman MB, Ideker T, Abate C, Jouvenet N, Kochs G, Shoichet B, Ott M, Palmarini M, Shokat KM, García-Sastre A, Rassen JA, Grosse R, Rosenberg OS, Verba KA, Basler CF, Vignuzzi M, Peden AA, Beltrao P, and Krogan NJ
- Subjects
- Conserved Sequence, Coronavirus Nucleocapsid Proteins genetics, Cryoelectron Microscopy, Humans, Mitochondrial Membrane Transport Proteins genetics, Mitochondrial Precursor Protein Import Complex Proteins, Phosphoproteins genetics, Phosphoproteins metabolism, Protein Conformation, COVID-19 metabolism, Coronavirus Nucleocapsid Proteins metabolism, Host Microbial Interactions, Mitochondrial Membrane Transport Proteins metabolism, Protein Interaction Maps, Severe acute respiratory syndrome-related coronavirus metabolism, SARS-CoV-2 metabolism, Severe Acute Respiratory Syndrome metabolism
- Abstract
The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2020
- Full Text
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9. Synthetic group A streptogramin antibiotics that overcome Vat resistance.
- Author
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Li Q, Pellegrino J, Lee DJ, Tran AA, Chaires HA, Wang R, Park JE, Ji K, Chow D, Zhang N, Brilot AF, Biel JT, van Zundert G, Borrelli K, Shinabarger D, Wolfe C, Murray B, Jacobson MP, Mühle E, Chesneau O, Fraser JS, and Seiple IB
- Subjects
- Acetylation drug effects, Acetyltransferases genetics, Acetyltransferases metabolism, Animals, Anti-Bacterial Agents classification, Bacterial Load drug effects, Binding Sites, Cryoelectron Microscopy, Female, In Vitro Techniques, Mice, Microbial Sensitivity Tests, Models, Molecular, RNA, Transfer metabolism, Ribosomes drug effects, Ribosomes metabolism, Staphylococcus aureus drug effects, Staphylococcus aureus genetics, Staphylococcus aureus metabolism, Streptogramin Group A chemistry, Streptogramin Group A classification, Virginiamycin analogs & derivatives, Virginiamycin chemistry, Virginiamycin metabolism, Anti-Bacterial Agents chemical synthesis, Anti-Bacterial Agents pharmacology, Drug Design, Drug Resistance, Bacterial drug effects, Streptogramin Group A chemical synthesis, Streptogramin Group A pharmacology
- Abstract
Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics
1 . Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2 , potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3 . Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2 . Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.- Published
- 2020
- Full Text
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10. An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation.
- Author
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Schoof M, Faust B, Saunders RA, Sangwan S, Rezelj V, Hoppe N, Boone M, Billesbølle CB, Puchades C, Azumaya CM, Kratochvil HT, Zimanyi M, Deshpande I, Liang J, Dickinson S, Nguyen HC, Chio CM, Merz GE, Thompson MC, Diwanji D, Schaefer K, Anand AA, Dobzinski N, Zha BS, Simoneau CR, Leon K, White KM, Chio US, Gupta M, Jin M, Li F, Liu Y, Zhang K, Bulkley D, Sun M, Smith AM, Rizo AN, Moss F, Brilot AF, Pourmal S, Trenker R, Pospiech T, Gupta S, Barsi-Rhyne B, Belyy V, Barile-Hill AW, Nock S, Liu Y, Krogan NJ, Ralston CY, Swaney DL, García-Sastre A, Ott M, Vignuzzi M, Walter P, and Manglik A
- Abstract
Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century., Competing Interests: Competing Interests M.Schoof, B.Faust, R.A.Saunders, N.Hoppe, P.Walter, and A.Manglik are inventors on a provisional patent describing anti-Spike nanobodies described in this manuscript.
- Published
- 2020
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11. Assembly of a nucleus-like structure during viral replication in bacteria.
- Author
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Chaikeeratisak V, Nguyen K, Khanna K, Brilot AF, Erb ML, Coker JK, Vavilina A, Newton GL, Buschauer R, Pogliano K, Villa E, Agard DA, and Pogliano J
- Subjects
- Capsid metabolism, Capsid Proteins biosynthesis, Capsid Proteins genetics, Cryoelectron Microscopy, Cytoplasm ultrastructure, Cytoplasm virology, DNA, Viral biosynthesis, Microscopy, Fluorescence, Pseudomonas Phages genetics, Pseudomonas chlororaphis ultrastructure, Transcription, Genetic, Pseudomonas Phages physiology, Pseudomonas chlororaphis virology, Virus Assembly
- Abstract
We observed the assembly of a nucleus-like structure in bacteria during viral infection. Using fluorescence microscopy and cryo-electron tomography, we showed that Pseudomonas chlororaphis phage 201φ2-1 assembled a compartment that separated viral DNA from the cytoplasm. The phage compartment was centered by a bipolar tubulin-based spindle, and it segregated phage and bacterial proteins according to function. Proteins involved in DNA replication and transcription localized inside the compartment, whereas proteins involved in translation and nucleotide synthesis localized outside. Later during infection, viral capsids assembled on the cytoplasmic membrane and moved to the surface of the compartment for DNA packaging. Ultimately, viral particles were released from the compartment and the cell lysed. These results demonstrate that phages have evolved a specialized structure to compartmentalize viral replication., (Copyright © 2017, American Association for the Advancement of Science.)
- Published
- 2017
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12. Ribosome•RelA structures reveal the mechanism of stringent response activation.
- Author
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Loveland AB, Bah E, Madireddy R, Zhang Y, Brilot AF, Grigorieff N, and Korostelev AA
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- Cryoelectron Microscopy, Protein Binding, Stress, Physiological, Escherichia coli physiology, Ligases metabolism, Ligases ultrastructure, RNA, Transfer metabolism, RNA, Transfer ultrastructure, Ribosomes metabolism, Ribosomes ultrastructure
- Abstract
Stringent response is a conserved bacterial stress response underlying virulence and antibiotic resistance. RelA/SpoT-homolog proteins synthesize transcriptional modulators (p)ppGpp, allowing bacteria to adapt to stress. RelA is activated during amino-acid starvation, when cognate deacyl-tRNA binds to the ribosomal A (aminoacyl-tRNA) site. We report four cryo-EM structures of E. coli RelA bound to the 70S ribosome, in the absence and presence of deacyl-tRNA accommodating in the 30S A site. The boomerang-shaped RelA with a wingspan of more than 100 Å wraps around the A/R (30S A-site/RelA-bound) tRNA. The CCA end of the A/R tRNA pins the central TGS domain against the 30S subunit, presenting the (p)ppGpp-synthetase domain near the 30S spur. The ribosome and A/R tRNA are captured in three conformations, revealing hitherto elusive states of tRNA engagement with the ribosomal decoding center. Decoding-center rearrangements are coupled with the step-wise 30S-subunit 'closure', providing insights into the dynamics of high-fidelity tRNA decoding.
- Published
- 2016
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13. Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction.
- Author
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Laxmikanthan G, Xu C, Brilot AF, Warren D, Steele L, Seah N, Tong W, Grigorieff N, Landy A, and Van Duyne GD
- Subjects
- Cryoelectron Microscopy, DNA, Bacterial genetics, DNA, Bacterial metabolism, DNA, Viral genetics, DNA, Viral metabolism, Imaging, Three-Dimensional, Models, Molecular, Bacteriophage lambda genetics, DNA, Bacterial chemistry, DNA, Cruciform chemistry, DNA, Viral chemistry, Escherichia coli genetics, Escherichia coli Proteins chemistry, Recombination, Genetic
- Abstract
The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation.
- Published
- 2016
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14. Evaluation of super-resolution performance of the K2 electron-counting camera using 2D crystals of aquaporin-0.
- Author
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Chiu PL, Li X, Li Z, Beckett B, Brilot AF, Grigorieff N, Agard DA, Cheng Y, and Walz T
- Subjects
- Animals, Cryoelectron Microscopy instrumentation, Crystallization, Crystallography, X-Ray, Electrons, Limit of Detection, Sheep, Aquaporins chemistry, Cryoelectron Microscopy methods, Eye Proteins chemistry, Image Processing, Computer-Assisted methods, Lens Capsule, Crystalline, Membrane Proteins analysis
- Abstract
The K2 Summit camera was initially the only commercially available direct electron detection camera that was optimized for high-speed counting of primary electrons and was also the only one that implemented centroiding so that the resolution of the camera can be extended beyond the Nyquist limit set by the physical pixel size. In this study, we used well-characterized two-dimensional crystals of the membrane protein aquaporin-0 to characterize the performance of the camera below and beyond the physical Nyquist limit and to measure the influence of electron dose rate on image amplitudes and phases., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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15. Structures of yeast 80S ribosome-tRNA complexes in the rotated and nonrotated conformations.
- Author
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Svidritskiy E, Brilot AF, Koh CS, Grigorieff N, and Korostelev AA
- Subjects
- Cryoelectron Microscopy, Image Processing, Computer-Assisted, Protein Biosynthesis genetics, RNA, Transfer metabolism, Ribosomes metabolism, Models, Molecular, Molecular Conformation, RNA, Transfer chemistry, Ribosomes chemistry, Saccharomyces cerevisiae Proteins chemistry
- Abstract
The structural understanding of eukaryotic translation lags behind that of translation on bacterial ribosomes. Here, we present two subnanometer resolution structures of S. cerevisiae 80S ribosome complexes formed with either one or two tRNAs and bound in response to an mRNA fragment containing the Kozak consensus sequence. The ribosomes adopt two globally different conformations that are related to each other by the rotation of the small subunit. Comparison with bacterial ribosome complexes reveals that the global structures and modes of intersubunit rotation of the yeast ribosome differ significantly from those in the bacterial counterpart, most notably in the regions involving the tRNA, small ribosomal subunit, and conserved helix 69 of the large ribosomal subunit. The structures provide insight into ribosome dynamics implicated in tRNA translocation and help elucidate the role of the Kozak fragment in positioning an open reading frame during translation initiation in eukaryotes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
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16. Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA-like structural element in the ribosomal decoding center.
- Author
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Koh CS, Brilot AF, Grigorieff N, and Korostelev AA
- Subjects
- Open Reading Frames, Picornaviridae genetics, RNA, Messenger genetics, RNA, Transfer genetics, RNA, Viral genetics, Ribosomes genetics, Nucleic Acid Conformation, Peptide Chain Initiation, Translational, Picornaviridae metabolism, RNA, Messenger metabolism, RNA, Transfer metabolism, RNA, Viral metabolism, Ribosomes metabolism
- Abstract
In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a cap-independent manner. We report two structures of the ribosome-bound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA-like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.
- Published
- 2014
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17. Cryo-EM near-atomic structure of a dsRNA fungal virus shows ancient structural motifs preserved in the dsRNA viral lineage.
- Author
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Luque D, Gómez-Blanco J, Garriga D, Brilot AF, González JM, Havens WM, Carrascosa JL, Trus BL, Verdaguer N, Ghabrial SA, and Castón JR
- Subjects
- Amino Acid Sequence, Capsid Proteins ultrastructure, Cryoelectron Microscopy, Molecular Sequence Data, Protein Folding, Protein Structure, Tertiary, RNA Viruses genetics, RNA, Double-Stranded genetics, Sequence Analysis, RNA, Evolution, Molecular, Models, Molecular, Nucleic Acid Conformation, Penicillium chrysogenum virology, RNA Viruses ultrastructure, RNA, Double-Stranded ultrastructure
- Abstract
Viruses evolve so rapidly that sequence-based comparison is not suitable for detecting relatedness among distant viruses. Structure-based comparisons suggest that evolution led to a small number of viral classes or lineages that can be grouped by capsid protein (CP) folds. Here, we report that the CP structure of the fungal dsRNA Penicillium chrysogenum virus (PcV) shows the progenitor fold of the dsRNA virus lineage and suggests a relationship between lineages. Cryo-EM structure at near-atomic resolution showed that the 982-aa PcV CP is formed by a repeated α-helical core, indicative of gene duplication despite lack of sequence similarity between the two halves. Superimposition of secondary structure elements identified a single "hotspot" at which variation is introduced by insertion of peptide segments. Structural comparison of PcV and other distantly related dsRNA viruses detected preferential insertion sites at which the complexity of the conserved α-helical core, made up of ancestral structural motifs that have acted as a skeleton, might have increased, leading to evolution of the highly varied current structures. Analyses of structural motifs only apparent after systematic structural comparisons indicated that the hallmark fold preserved in the dsRNA virus lineage shares a long (spinal) α-helix tangential to the capsid surface with the head-tailed phage and herpesvirus viral lineage.
- Published
- 2014
- Full Text
- View/download PDF
18. Structure of the ribosome with elongation factor G trapped in the pretranslocation state.
- Author
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Brilot AF, Korostelev AA, Ermolenko DN, and Grigorieff N
- Subjects
- Cryoelectron Microscopy, Models, Molecular, Nucleic Acid Conformation, Peptide Elongation Factor G chemistry, Protein Biosynthesis physiology, Ribosomes chemistry
- Abstract
During protein synthesis, tRNAs and their associated mRNA codons move sequentially on the ribosome from the A (aminoacyl) site to the P (peptidyl) site to the E (exit) site in a process catalyzed by a universally conserved ribosome-dependent GTPase [elongation factor G (EF-G) in prokaryotes and elongation factor 2 (EF-2) in eukaryotes]. Although the high-resolution structure of EF-G bound to the posttranslocation ribosome has been determined, the pretranslocation conformation of the ribosome bound with EF-G and A-site tRNA has evaded visualization owing to the transient nature of this state. Here we use electron cryomicroscopy to determine the structure of the 70S ribosome with EF-G, which is trapped in the pretranslocation state using antibiotic viomycin. Comparison with the posttranslocation ribosome shows that the small subunit of the pretranslocation ribosome is rotated by ∼12° relative to the large subunit. Domain IV of EF-G is positioned in the cleft between the body and head of the small subunit outwardly of the A site and contacts the A-site tRNA. Our findings suggest a model in which domain IV of EF-G promotes the translocation of tRNA from the A to the P site as the small ribosome subunit spontaneously rotates back from the hybrid, rotated state into the nonrotated posttranslocation state.
- Published
- 2013
- Full Text
- View/download PDF
19. Likelihood-based classification of cryo-EM images using FREALIGN.
- Author
-
Lyumkis D, Brilot AF, Theobald DL, and Grigorieff N
- Subjects
- Algorithms, Bayes Theorem, Computer Simulation, Cryoelectron Microscopy methods, Escherichia coli, Likelihood Functions, Models, Molecular, Ribosome Subunits, Large, Bacterial ultrastructure, Ribosome Subunits, Small, Bacterial ultrastructure, Image Processing, Computer-Assisted, Software
- Abstract
We describe an implementation of maximum likelihood classification for single particle electron cryo-microscopy that is based on the FREALIGN software. Particle alignment parameters are determined by maximizing a joint likelihood that can include hierarchical priors, while classification is performed by expectation maximization of a marginal likelihood. We test the FREALIGN implementation using a simulated dataset containing computer-generated projection images of three different 70S ribosome structures, as well as a publicly available dataset of 70S ribosomes. The results show that the mixed strategy of the new FREALIGN algorithm yields performance on par with other maximum likelihood implementations, while remaining computationally efficient., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
20. Movies of ice-embedded particles enhance resolution in electron cryo-microscopy.
- Author
-
Campbell MG, Cheng A, Brilot AF, Moeller A, Lyumkis D, Veesler D, Pan J, Harrison SC, Potter CS, Carragher B, and Grigorieff N
- Subjects
- Cryoelectron Microscopy methods, Ice, Rotavirus ultrastructure
- Abstract
Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We show here that we can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities. This procedure increases the efficiency of cryo-EM imaging and enhances the resolution obtained in three-dimensional reconstructions of the particle. Using movies in this way is generally applicable to all cryo-EM samples and should also improve the performance of midrange electron microscopes that may have limited mechanical stability and beam coherence., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
21. Beam-induced motion of vitrified specimen on holey carbon film.
- Author
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Brilot AF, Chen JZ, Cheng A, Pan J, Harrison SC, Potter CS, Carragher B, Henderson R, and Grigorieff N
- Subjects
- Image Processing, Computer-Assisted methods, Cryoelectron Microscopy methods
- Abstract
The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
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