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1.
bioRxiv ; 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38559232

RESUMO

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAP), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, time-resolved cryo-electron microscopy (cryo-EM) was used to capture four intermediates populated 120 or 500 milliseconds (ms) after mixing Escherichia coli σ70-RNAP and the λPR promoter. Cryo-EM snapshots revealed the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As nt-strand "read-out" extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating yet unknown conformational changes load it in subsequent steps. Because these events likely describe DNA opening at many bacterial promoters, this study provides needed insights into how DNA sequence regulates steps of RPo formation.

2.
Nucleic Acids Res ; 52(2): 831-843, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38084901

RESUMO

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.


Assuntos
Empacotamento do Genoma Viral , Montagem de Vírus , Montagem de Vírus/genética , Bacteriófago lambda/genética , Endodesoxirribonucleases/metabolismo , DNA , DNA Viral/metabolismo , Empacotamento do DNA
4.
Microsc Microanal ; 29(29 Suppl 1): 949-950, 2023 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-37613800
5.
IUCrJ ; 10(Pt 1): 77-89, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36598504

RESUMO

Single-particle cryo-electron microscopy (cryoEM) is a swiftly growing method for understanding protein structure. With increasing demand for high-throughput, high-resolution cryoEM services comes greater demand for rapid and automated cryoEM grid and sample screening. During screening, optimal grids and sample conditions are identified for subsequent high-resolution data collection. Screening is a major bottleneck for new cryoEM projects because grids must be optimized for several factors, including grid type, grid hole size, sample concentration, buffer conditions, ice thickness and particle behavior. Even for mature projects, multiple grids are commonly screened to select a subset for high-resolution data collection. Here, machine learning and novel purpose-built image-processing and microscope-handling algorithms are incorporated into the automated data-collection software Leginon, to provide an open-source solution for fully automated high-throughput grid screening. This new version, broadly called Smart Leginon, emulates the actions of an operator in identifying areas on the grid to explore as potentially useful for data collection. Smart Leginon Autoscreen sequentially loads and examines grids from an automated specimen-exchange system to provide completely unattended grid screening across a set of grids. Comparisons between a multi-grid autoscreen session and conventional manual screening by 5 expert microscope operators are presented. On average, Autoscreen reduces operator time from ∼6 h to <10 min and provides a percentage of suitable images for evaluation comparable to the best operator. The ability of Smart Leginon to target holes that are particularly difficult to identify is analyzed. Finally, the utility of Smart Leginon is illustrated with three real-world multi-grid user screening/collection sessions, demonstrating the efficiency and flexibility of the software package. The fully automated functionality of Smart Leginon significantly reduces the burden on operator screening time, improves the throughput of screening and recovers idle microscope time, thereby improving availability of cryoEM services.


Assuntos
Processamento de Imagem Assistida por Computador , Software , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Elétrons
6.
Annu Rev Biochem ; 91: 1-32, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35320683

RESUMO

Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.


Assuntos
COVID-19 , Pandemias , Microscopia Crioeletrônica/métodos , Humanos , SARS-CoV-2 , Imagem Individual de Molécula
7.
Nat Struct Mol Biol ; 29(3): 250-260, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35260847

RESUMO

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex or RTC) associated with two copies of nsp13 (nsp132-RTC). One copy of nsp13 interacts with the template-RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here we use cryogenic-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results indicate a mechanism for the nsp132-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.


Assuntos
COVID-19 , SARS-CoV-2 , Microscopia Crioeletrônica , Humanos , RNA Helicases/química , Proteínas não Estruturais Virais/química , Replicação Viral
8.
Cell ; 182(6): 1560-1573.e13, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32783916

RESUMO

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.


Assuntos
Metiltransferases/química , RNA Helicases/química , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química , Replicação Viral , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , RNA-Polimerase RNA-Dependente de Coronavírus , Microscopia Crioeletrônica , Holoenzimas/química , Holoenzimas/metabolismo , Magnésio/metabolismo , Metiltransferases/metabolismo , Ligação Proteica , RNA Helicases/metabolismo , RNA Viral/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2 , Proteínas não Estruturais Virais/metabolismo
9.
Nat Methods ; 17(9): 897-900, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32778833

RESUMO

We present an approach for preparing cryo-electron microscopy (cryo-EM) grids to study short-lived molecular states. Using piezoelectric dispensing, two independent streams of ~50-pl droplets of sample are deposited within 10 ms of each other onto the surface of a nanowire EM grid, and the mixing reaction stops when the grid is vitrified in liquid ethane ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.


Assuntos
Microscopia Crioeletrônica/métodos , Nanofios , Robótica , Manejo de Espécimes/métodos , Fatores de Tempo
10.
Structure ; 28(7): 727-729, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32640251

RESUMO

In this issue of Structure, Li et al. (2020) describe machine learning methods that automate decision making in the cryo-EM pre-processing pipeline, eliminating the need for user-subjective decisions. They successfully tested this pipeline for a wide range of datasets, demonstrating the proof of concept and the efficiency of this approach.


Assuntos
Tomada de Decisões , Microscopia Crioeletrônica
11.
bioRxiv ; 2020 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-32676607

RESUMO

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.

12.
Nat Commun ; 10(1): 4521, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586061

RESUMO

Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules.


Assuntos
Anticorpos/química , Domínios PDZ , Peptídeos/química , Engenharia de Proteínas , Mapas de Interação de Proteínas/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Células COS , Chlorocebus aethiops , Proteína 4 Homóloga a Disks-Large/antagonistas & inibidores , Proteína 4 Homóloga a Disks-Large/metabolismo , Desenho de Fármacos , Mapeamento de Epitopos , Modelos Moleculares , Biblioteca de Peptídeos , Peptídeos/farmacologia , Ligação Proteica , Proteínas Recombinantes/metabolismo
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