RESUMEN
The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of ß-tubulin using human prefoldin and TRiC. We find unstructured ß-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded ß-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.
Asunto(s)
Chaperonina con TCP-1 , Tubulina (Proteína) , Humanos , Chaperonina con TCP-1/química , Tubulina (Proteína)/metabolismo , Pliegue de Proteína , Proteostasis , Adenosina Trifosfato/metabolismoRESUMEN
Maintaining proteostasis in eukaryotic protein folding involves cooperation of distinct chaperone systems. To understand how the essential ring-shaped chaperonin TRiC/CCT cooperates with the chaperone prefoldin/GIMc (PFD), we integrate cryoelectron microscopy (cryo-EM), crosslinking-mass-spectrometry and biochemical and cellular approaches to elucidate the structural and functional interplay between TRiC/CCT and PFD. We find these hetero-oligomeric chaperones associate in a defined architecture, through a conserved interface of electrostatic contacts that serves as a pivot point for a TRiC-PFD conformational cycle. PFD alternates between an open "latched" conformation and a closed "engaged" conformation that aligns the PFD-TRiC substrate binding chambers. PFD can act after TRiC bound its substrates to enhance the rate and yield of the folding reaction, suppressing non-productive reaction cycles. Disrupting the TRiC-PFD interaction in vivo is strongly deleterious, leading to accumulation of amyloid aggregates. The supra-chaperone assembly formed by PFD and TRiC is essential to prevent toxic conformations and ensure effective cellular proteostasis.
Asunto(s)
Chaperonina con TCP-1/metabolismo , Chaperonas Moleculares/metabolismo , Proteostasis/fisiología , Actinas/química , Actinas/metabolismo , Chaperonina con TCP-1/química , Chaperonina con TCP-1/genética , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , Electricidad EstáticaRESUMEN
Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.
Asunto(s)
Microscopía por Crioelectrón , Ebolavirus/fisiología , Ebolavirus/ultraestructura , Nucleocápside/ultraestructura , Nucleoproteínas/ultraestructura , Ensamble de Virus , Modelos Biológicos , Proteínas Mutantes/química , Mutación/genética , Nucleoproteínas/química , Multimerización de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN Viral/biosíntesis , ARN Viral/química , ARN Viral/metabolismoRESUMEN
The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein-nucleic acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: Escherichia coli beta-galactosidase with inhibitor, SARS-CoV-2 virus RNA-dependent RNA polymerase with covalently bound nucleotide analog and SARS-CoV-2 virus ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. The quality of submitted ligand models and surrounding atoms were analyzed by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics and contact scores. A composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.
Asunto(s)
Microscopía por Crioelectrón , Modelos Moleculares , Microscopía por Crioelectrón/métodos , Ligandos , SARS-CoV-2 , COVID-19/virología , Escherichia coli , beta-Galactosidasa/química , beta-Galactosidasa/metabolismo , Conformación Proteica , Reproducibilidad de los ResultadosRESUMEN
Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution1-3. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships4, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes.
Asunto(s)
Microscopía por Crioelectrón , Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Catalítico/ultraestructura , Tetrahymena thermophila , Apoenzimas/química , Apoenzimas/ultraestructura , Holoenzimas/química , Holoenzimas/ultraestructura , Modelos Moleculares , Tetrahymena thermophila/enzimología , Tetrahymena thermophila/genéticaRESUMEN
The molecular mechanism of transmembrane proton translocation in rotary motor ATPases is not fully understood. Here, we report the 3.5-Å resolution cryoEM structure of the lipid nanodisc-reconstituted Vo proton channel of the yeast vacuolar H+-ATPase, captured in a physiologically relevant, autoinhibited state. The resulting atomic model provides structural detail for the amino acids that constitute the proton pathway at the interface of the proteolipid ring and subunit a. Based on the structure and previous mutagenesis studies, we propose the chemical basis of transmembrane proton transport. Moreover, we discovered that the C terminus of the assembly factor Voa1 is an integral component of mature Vo. Voa1's C-terminal transmembrane α helix is bound inside the proteolipid ring, where it contributes to the stability of the complex. Our structure rationalizes possible mechanisms by which mutations in human Vo can result in disease phenotypes and may thus provide new avenues for therapeutic interventions.
Asunto(s)
Microscopía por Crioelectrón , Nanopartículas , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/enzimología , ATPasas de Translocación de Protón Vacuolares/ultraestructura , Genotipo , Humanos , Lípidos de la Membrana/química , Modelos Moleculares , Mutación , Fenotipo , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína , Protones , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Nuclear receptors recruit multiple coactivators sequentially to activate transcription. This "ordered" recruitment allows different coactivator activities to engage the nuclear receptor complex at different steps of transcription. Estrogen receptor (ER) recruits steroid receptor coactivator-3 (SRC-3) primary coactivator and secondary coactivators, p300/CBP and CARM1. CARM1 recruitment lags behind the binding of SRC-3 and p300 to ER. Combining cryo-electron microscopy (cryo-EM) structure analysis and biochemical approaches, we demonstrate that there is a close crosstalk between early- and late-recruited coactivators. The sequential recruitment of CARM1 not only adds a protein arginine methyltransferase activity to the ER-coactivator complex, it also alters the structural organization of the pre-existing ERE/ERα/SRC-3/p300 complex. It induces a p300 conformational change and significantly increases p300 HAT activity on histone H3K18 residues, which, in turn, promotes CARM1 methylation activity on H3R17 residues to enhance transcriptional activity. This study reveals a structural role for a coactivator sequential recruitment and biochemical process in ER-mediated transcription.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Receptor alfa de Estrógeno/metabolismo , Guanilato Ciclasa/metabolismo , Coactivador 3 de Receptor Nuclear/metabolismo , Transcripción Genética , Acetilación , Sitios de Unión , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/genética , Microscopía por Crioelectrón , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/genética , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/genética , Guanilato Ciclasa/química , Guanilato Ciclasa/genética , Células HEK293 , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Células MCF-7 , Metilación , Modelos Moleculares , Complejos Multiproteicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Coactivador 3 de Receptor Nuclear/química , Coactivador 3 de Receptor Nuclear/genética , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Factores de Tiempo , Factores de Transcripción , Activación Transcripcional , TransfecciónRESUMEN
Tetrahymena ribozyme is a group I intron, whose self-splicing is the result of two sequential ester-transfer reactions. To understand how it facilitates catalysis in the first self-splicing reaction, we used cryogenic electron microscopy (cryo-EM) to resolve the structures of L-16 Tetrahymena ribozyme complexed with a 11-nucleotide 5'-splice site analog substrate. Four conformations were achieved to 4.14, 3.18, 3.09 and 2.98 Å resolutions, respectively, corresponding to different splicing intermediates during the first enzymatic reaction. Comparison of these structures reveals structural alterations, including large conformational changes in IGS/IGSext (P1-P1ext duplex) and J5/4, as well as subtle local rearrangements in the G-binding site. These structural changes are required for the enzymatic activity of the Tetrahymena ribozyme. Our study demonstrates the ability of cryo-EM to capture dynamic RNA structural changes, ushering in a new era in the analysis of RNA structure-function by cryo-EM.
Asunto(s)
ARN Catalítico , Tetrahymena , Secuencia de Bases , Microscopía por Crioelectrón , Intrones , Conformación de Ácido Nucleico , ARN , Empalme del ARN , ARN Catalítico/química , ARN Catalítico/ultraestructuraRESUMEN
The Tetrahymena group I intron has been a key system in the understanding of RNA folding and misfolding. The molecule folds into a long-lived misfolded intermediate (M) in vitro, which has been known to form extensive native-like secondary and tertiary structures but is separated by an unknown kinetic barrier from the native state (N). Here, we used cryogenic electron microscopy (cryo-EM) to resolve misfolded structures of the Tetrahymena L-21 ScaI ribozyme. Maps of three M substates (M1, M2, M3) and one N state were achieved from a single specimen with overall resolutions of 3.5 Å, 3.8 Å, 4.0 Å, and 3.0 Å, respectively. Comparisons of the structures reveal that all the M substates are highly similar to N, except for rotation of a core helix P7 that harbors the ribozyme's guanosine binding site and the crossing of the strands J7/3 and J8/7 that connect P7 to the other elements in the ribozyme core. This topological difference between the M substates and N state explains the failure of 5'-splice site substrate docking in M, supports a topological isomer model for the slow refolding of M to N due to a trapped strand crossing, and suggests pathways for M-to-N refolding.
Asunto(s)
Pliegue del ARN , ARN Catalítico , Tetrahymena , Microscopía por Crioelectrón , Cinética , ARN Catalítico/química , Tetrahymena/genéticaRESUMEN
Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in Toxoplasma gondii, an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing cytoskeletal components are subpellicular microtubules (SPMTs) and conoid fibrils (CFs). The SPMTs help maintain shape and gliding motility, while the CFs are implicated in invasion. Here, we use cryogenic electron tomography to determine the molecular structures of the SPMTs and CFs in vitrified intact and detergent-extracted parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intralumenal spiral lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite's long axis. Each CF is composed of nine tubulin protofilaments that display a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism illustrates the versatility of tubulin to form two distinct types of assemblies, SPMTs and CFs.
Asunto(s)
Parásitos/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Citoesqueleto/metabolismo , Tomografía con Microscopio Electrónico/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Orgánulos/metabolismoRESUMEN
This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.
Asunto(s)
Microscopía por Crioelectrón/métodos , Modelos Moleculares , Cristalografía por Rayos X , Conformación Proteica , Proteínas/químicaRESUMEN
RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas , ARN/metabolismo , Ribosomas/metabolismo , Biología Sintética/métodosRESUMEN
Cryogenic electron microscopy (cryo-EM) maps are now at the point where resolvability of individual atoms can be achieved. However, resolvability is not necessarily uniform throughout the map. We introduce a quantitative parameter to characterize the resolvability of individual atoms in cryo-EM maps, the map Q-score. Q-scores can be calculated for atoms in proteins, nucleic acids, water, ligands and other solvent atoms, using models fitted to or derived from cryo-EM maps. Q-scores can also be averaged to represent larger features such as entire residues and nucleotides. Averaged over entire models, Q-scores correlate very well with the estimated resolution of cryo-EM maps for both protein and RNA. Assuming the models they are calculated from are well fitted to the map, Q-scores can be used as a measure of resolvability in cryo-EM maps at various scales, from entire macromolecules down to individual atoms. Q-score analysis of multiple cryo-EM maps of the same proteins derived from different laboratories confirms the reproducibility of structural features from side chains down to water and ion atoms.
Asunto(s)
Apoferritinas/química , Microscopía por Crioelectrón , Algoritmos , Animales , Análisis de Fourier , Humanos , Enlace de Hidrógeno , Ligandos , Sustancias Macromoleculares/química , Ratones , Microscopía Electrónica , Modelos Moleculares , Distribución Normal , Estructura Secundaria de Proteína , ARN/química , Solventes/químicaRESUMEN
The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.
Asunto(s)
Microscopía por Crioelectrón/métodos , ARN/química , Simulación por Computador , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Catalítico/química , RiboswitchRESUMEN
Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae, including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109KSQD112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109AAAA112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae.
Asunto(s)
Herpesvirus Humano 3/fisiología , Melanoma/metabolismo , Fusión de Membrana , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Melanoma/virología , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Dominios Proteicos , Homología de Secuencia , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Matrix proteins are encoded by many enveloped viruses, including influenza viruses, herpes viruses, and coronaviruses. Underneath the viral envelope of influenza virus, matrix protein 1 (M1) forms an oligomeric layer critical for particle stability and pH-dependent RNA genome release. However, high-resolution structures of full-length monomeric M1 and the matrix layer have not been available, impeding antiviral targeting and understanding of the pH-dependent transitions involved in cell entry. Here, purification and extensive mutagenesis revealed protein-protein interfaces required for the formation of multilayered helical M1 oligomers similar to those observed in virions exposed to the low pH of cell entry. However, single-layered helical oligomers with biochemical and ultrastructural similarity to those found in infectious virions before cell entry were observed upon mutation of a single amino acid. The highly ordered structure of the single-layered oligomers and their likeness to the matrix layer of intact virions prompted structural analysis by cryo-electron microscopy (cryo-EM). The resulting 3.4-Å-resolution structure revealed the molecular details of M1 folding and its organization within the single-shelled matrix. The solution of the full-length M1 structure, the identification of critical assembly interfaces, and the development of M1 assembly assays with purified proteins are crucial advances for antiviral targeting of influenza viruses.
Asunto(s)
Imagenología Tridimensional , Proteínas de la Matriz Viral/química , Secuencia de Aminoácidos , Reactivos de Enlaces Cruzados/química , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutación/genética , Multimerización de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Recombinación Genética/genética , Proteínas de la Matriz Viral/genética , Virión/ultraestructuraRESUMEN
Estrogen receptor (ER/ESR1) is a transcription factor critical for development, reproduction, metabolism, and cancer. ER function hinges on its ability to recruit primary and secondary coactivators, yet structural information on the full-length receptor-coactivator complex to complement preexisting and sometimes controversial biochemical information is lacking. Here, we use cryoelectron microscopy (cryo-EM) to determine the quaternary structure of an active complex of DNA-bound ERα, steroid receptor coactivator 3 (SRC-3/NCOA3), and a secondary coactivator (p300/EP300). Our structural model suggests the following assembly mechanism for the complex: each of the two ligand-bound ERα monomers independently recruits one SRC-3 protein via the transactivation domain of ERα; the two SRC-3s in turn bind to different regions of one p300 protein through multiple contacts. We also present structural evidence for the location of activation function 1 (AF-1) in a full-length nuclear receptor, which supports a role for AF-1 in SRC-3 recruitment.
Asunto(s)
Proteína p300 Asociada a E1A/química , Receptor alfa de Estrógeno/química , Coactivador 3 de Receptor Nuclear/química , Sitios de Unión , Microscopía por Crioelectrón , ADN/química , ADN/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Receptor alfa de Estrógeno/metabolismo , Histona Acetiltransferasas/metabolismo , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Coactivador 3 de Receptor Nuclear/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Elementos de RespuestaRESUMEN
Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR-Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR-Cas systems. The type I-F CRISPR-Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 Å, 3.42 Å, and 3.15 Å, respectively. The 2.57-Å structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR-Cas system.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Interacciones Huésped-Patógeno , Proteínas Virales/metabolismo , Proteínas Asociadas a CRISPR/ultraestructura , Sistemas CRISPR-Cas , Microscopía por Crioelectrón , Pseudomonas aeruginosa , Proteínas Virales/ultraestructuraRESUMEN
Cryogenic electron microscopy (cryo-EM) methods began to be used in the mid-1970s to study thin and periodic arrays of proteins. Following a half-century of development in cryo-specimen preparation, instrumentation, data collection, data processing, and modeling software, cryo-EM has become a routine method for solving structures from large biological assemblies to small biomolecules at near to true atomic resolution. This review explores the critical roles played by the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) in partnership with the community to develop the necessary infrastructure to archive cryo-EM maps and associated models. Public access to cryo-EM structure data has in turn facilitated better understanding of structure-function relationships and advancement of image processing and modeling tool development. The partnership between the global cryo-EM community and PDB and EMDB leadership has synergistically shaped the standards for metadata, one-stop deposition of maps and models, and validation metrics to assess the quality of cryo-EM structures. The advent of cryo-electron tomography (cryo-ET) for in situ molecular cell structures at a broad resolution range and their correlations with other imaging data introduce new data archival challenges in terms of data size and complexity in the years to come.