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1.
Science ; 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154106

RESUMO

The 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. Cryogenic electron microscopy (cryo-EM) revealed that one 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 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.

2.
Nature ; 586(7827): 145-150, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968273

RESUMO

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 antibiotics1. 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.

3.
Science ; 355(6321): 194-197, 2017 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-28082593

RESUMO

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.


Assuntos
Fagos de Pseudomonas/fisiologia , Pseudomonas chlororaphis/virologia , Montagem de Vírus , Capsídeo/metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Microscopia Crioeletrônica , Citoplasma/ultraestrutura , Citoplasma/virologia , DNA Viral/biossíntese , Microscopia de Fluorescência , Fagos de Pseudomonas/genética , Pseudomonas chlororaphis/ultraestrutura , Transcrição Genética
4.
Elife ; 52016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27434674

RESUMO

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.


Assuntos
Escherichia coli/fisiologia , Ligases/metabolismo , Ligases/ultraestrutura , RNA de Transferência/metabolismo , RNA de Transferência/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Microscopia Crioeletrônica , Ligação Proteica , Estresse Fisiológico
5.
Elife ; 52016 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-27223329

RESUMO

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.


Assuntos
Bacteriófago lambda/genética , DNA Bacteriano/química , DNA Cruciforme/química , DNA Viral/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Recombinação Genética , Microscopia Crioeletrônica , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Imageamento Tridimensional , Modelos Moleculares
6.
J Struct Biol ; 192(2): 163-73, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26318383

RESUMO

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.


Assuntos
Aquaporinas/química , Microscopia Crioeletrônica/métodos , Proteínas do Olho/química , Processamento de Imagem Assistida por Computador/métodos , Cápsula do Cristalino , Proteínas de Membrana/análise , Animais , Microscopia Crioeletrônica/instrumentação , Cristalização , Cristalografia por Raios X , Elétrons , Limite de Detecção , Ovinos
7.
Structure ; 22(8): 1210-1218, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-25043550

RESUMO

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.


Assuntos
Modelos Moleculares , Conformação Molecular , RNA de Transferência/química , Ribossomos/química , Proteínas de Saccharomyces cerevisiae/química , Microscopia Crioeletrônica , Processamento de Imagem Assistida por Computador , Biossíntese de Proteínas/genética , RNA de Transferência/metabolismo , Ribossomos/metabolismo
8.
Proc Natl Acad Sci U S A ; 111(25): 9139-44, 2014 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-24927574

RESUMO

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.


Assuntos
Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica , Picornaviridae/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , RNA Viral/metabolismo , Ribossomos/metabolismo , Fases de Leitura Aberta , Picornaviridae/genética , RNA Mensageiro/genética , RNA de Transferência/genética , RNA Viral/genética , Ribossomos/genética
9.
Proc Natl Acad Sci U S A ; 111(21): 7641-6, 2014 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-24821769

RESUMO

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.


Assuntos
Evolução Molecular , Modelos Moleculares , Conformação de Ácido Nucleico , Penicillium chrysogenum/virologia , Vírus de RNA/ultraestrutura , RNA de Cadeia Dupla/ultraestrutura , Sequência de Aminoácidos , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , Análise de Sequência de RNA
10.
Proc Natl Acad Sci U S A ; 110(52): 20994-9, 2013 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-24324137

RESUMO

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.


Assuntos
Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/química , Biossíntese de Proteínas/fisiologia , Ribossomos/química , Microscopia Crioeletrônica
11.
J Struct Biol ; 183(3): 377-388, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23872434

RESUMO

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.


Assuntos
Processamento de Imagem Assistida por Computador , Software , Algoritmos , Teorema de Bayes , Simulação por Computador , Microscopia Crioeletrônica/métodos , Escherichia coli , Funções Verossimilhança , Modelos Moleculares , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura
12.
Structure ; 20(11): 1823-8, 2012 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-23022349

RESUMO

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.


Assuntos
Microscopia Crioeletrônica/métodos , Gelo , Rotavirus/ultraestrutura
13.
J Struct Biol ; 177(3): 630-7, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22366277

RESUMO

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.


Assuntos
Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos
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