RESUMO
Accurately building 3D atomic structures from cryo-EM density maps is a crucial step in cryo-EM-based protein structure determination. Converting density maps into 3D atomic structures for proteins lacking accurate homologous or predicted structures as templates remains a significant challenge. Here, we introduce Cryo2Struct, a fully automated de novo cryo-EM structure modeling method. Cryo2Struct utilizes a 3D transformer to identify atoms and amino acid types in cryo-EM density maps, followed by an innovative Hidden Markov Model (HMM) to connect predicted atoms and build protein backbone structures. Cryo2Struct produces substantially more accurate and complete protein structural models than the widely used ab initio method Phenix. Additionally, its performance in building atomic structural models is robust against changes in the resolution of density maps and the size of protein structures.
Assuntos
Microscopia Crioeletrônica , Cadeias de Markov , Modelos Moleculares , Conformação Proteica , Proteínas , Microscopia Crioeletrônica/métodos , Proteínas/química , Proteínas/ultraestrutura , Algoritmos , SoftwareRESUMO
Converting cryo-electron microscopy (cryo-EM) data into high-quality structural models is a challenging problem of outstanding importance. Current refinement methods often generate unbalanced models in which physico-chemical quality is sacrificed for excellent fit to the data. Furthermore, these techniques struggle to represent the conformational heterogeneity averaged out in low-resolution regions of density maps. Here we introduce EMMIVox, a Bayesian inference approach to determine single-structure models as well as structural ensembles from cryo-EM maps. EMMIVox automatically balances experimental information with accurate physico-chemical models of the system and the surrounding environment, including waters, lipids, and ions. Explicit treatment of data correlation and noise as well as inference of accurate B-factors enable determination of structural models and ensembles with both excellent fit to the data and high stereochemical quality, thus outperforming state-of-the-art refinement techniques. EMMIVox represents a flexible approach to determine high-quality structural models that will contribute to advancing our understanding of the molecular mechanisms underlying biological functions.
Assuntos
Teorema de Bayes , Microscopia Crioeletrônica , Modelos Moleculares , Microscopia Crioeletrônica/métodos , Biologia Computacional/métodos , Conformação Proteica , Algoritmos , Proteínas/química , Proteínas/ultraestruturaRESUMO
The advent of single-particle cryo-electron microscopy (cryo-EM) has brought forth a new era of structural biology, enabling the routine determination of large biological molecules and their complexes at atomic resolution. The high-resolution structures of biological macromolecules and their complexes significantly expedite biomedical research and drug discovery. However, automatically and accurately building atomic models from high-resolution cryo-EM density maps is still time-consuming and challenging when template-based models are unavailable. Artificial intelligence (AI) methods such as deep learning trained on limited amount of labeled cryo-EM density maps generate inaccurate atomic models. To address this issue, we created a dataset called Cryo2StructData consisting of 7,600 preprocessed cryo-EM density maps whose voxels are labelled according to their corresponding known atomic structures for training and testing AI methods to build atomic models from cryo-EM density maps. Cryo2StructData is larger than existing, publicly available datasets for training AI methods to build atomic protein structures from cryo-EM density maps. We trained and tested deep learning models on Cryo2StructData to validate its quality showing that it is ready for being used to train and test AI methods for building atomic models.
Assuntos
Inteligência Artificial , Microscopia Crioeletrônica , Proteínas , Microscopia Crioeletrônica/métodos , Proteínas/química , Proteínas/ultraestrutura , Modelos Moleculares , Conformação ProteicaRESUMO
Proteins often undergo large-scale conformational transitions, in which secondary and tertiary structure elements (loops, helices, and domains) change their structures or their positions with respect to each other. Simple considerations suggest that such dynamics should be relatively fast, but the functional cycles of many proteins are often relatively slow. Sophisticated experimental methods are starting to tackle this dichotomy and shed light on the contribution of large-scale conformational dynamics to protein function. In this review, we focus on the contribution of single-molecule Förster resonance energy transfer and nuclear magnetic resonance (NMR) spectroscopies to the study of conformational dynamics. We briefly describe the state of the art in each of these techniques and then point out their similarities and differences, as well as the relative strengths and weaknesses of each. Several case studies, in which the connection between fast conformational dynamics and slower function has been demonstrated, are then introduced and discussed. These examples include both enzymes and large protein machines, some of which have been studied by both NMR and fluorescence spectroscopies.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/química , Proteínas/metabolismo , Proteínas/ultraestrutura , Conformação Proteica , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Imagem Individual de Molécula/métodos , Movimento (Física)RESUMO
Interpreting electron cryo-microscopy (cryo-EM) maps with atomic models requires high levels of expertise and labour-intensive manual intervention in three-dimensional computer graphics programs1,2. Here we present ModelAngelo, a machine-learning approach for automated atomic model building in cryo-EM maps. By combining information from the cryo-EM map with information from protein sequence and structure in a single graph neural network, ModelAngelo builds atomic models for proteins that are of similar quality to those generated by human experts. For nucleotides, ModelAngelo builds backbones with similar accuracy to those built by humans. By using its predicted amino acid probabilities for each residue in hidden Markov model sequence searches, ModelAngelo outperforms human experts in the identification of proteins with unknown sequences. ModelAngelo will therefore remove bottlenecks and increase objectivity in cryo-EM structure determination.
Assuntos
Microscopia Crioeletrônica , Aprendizado de Máquina , Modelos Moleculares , Proteínas , Sequência de Aminoácidos , Microscopia Crioeletrônica/métodos , Microscopia Crioeletrônica/normas , Cadeias de Markov , Redes Neurais de Computação , Conformação Proteica , Proteínas/química , Proteínas/ultraestrutura , Gráficos por ComputadorAssuntos
Métodos Analíticos de Preparação de Amostras , Microscopia Crioeletrônica , Espectrometria de Massas , Proteínas , Biologia Molecular/métodos , Biologia Molecular/tendências , Microscopia Crioeletrônica/métodos , Microscopia Crioeletrônica/normas , Microscopia Crioeletrônica/tendências , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Espectrometria de Massas/tendências , Proteínas/química , Proteínas/ultraestrutura , Métodos Analíticos de Preparação de Amostras/métodos , Métodos Analíticos de Preparação de Amostras/normasRESUMO
There has been considerable recent progress in designing new proteins using deep-learning methods1-9. Despite this progress, a general deep-learning framework for protein design that enables solution of a wide range of design challenges, including de novo binder design and design of higher-order symmetric architectures, has yet to be described. Diffusion models10,11 have had considerable success in image and language generative modelling but limited success when applied to protein modelling, probably due to the complexity of protein backbone geometry and sequence-structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topology-constrained protein monomer design, protein binder design, symmetric oligomer design, enzyme active site scaffolding and symmetric motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by experimentally characterizing the structures and functions of hundreds of designed symmetric assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple molecular specifications.
Assuntos
Aprendizado Profundo , Proteínas , Domínio Catalítico , Microscopia Crioeletrônica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/ultraestrutura , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Proteínas/ultraestruturaRESUMO
Ice thickness is arguably one of the most important factors limiting the resolution of protein structures determined by cryo-electron microscopy (cryo-EM). The amorphous atomic structure of the ice that stabilizes and protects biological samples in cryo-EM grids also imprints some additional noise in cryo-EM images. Ice that is too thick jeopardizes the success of particle picking and reconstruction of the biomolecule in the worst case and, at best, deteriorates eventual map resolution. Minimizing the thickness of the ice layer and thus the magnitude of its noise contribution is thus imperative in cryo-EM grid preparation. In this paper we introduce MeasureIce, a simple, easy to use ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. We show that it is possible to simulate thickness-image intensity look-up tables, also usable in SerialEM and Leginon, using elementary scattering physics and thereby adapt the tool to any microscope without time consuming experimental calibration. We benchmark our approach using two alternative techniques: the "ice channel" technique and tilt-series tomography. We also demonstrate the utility of ice thickness measurement for selecting holes in gold grids containing an Equine apoferritin sample, achieving a 1.88 Ångstrom resolution in subsequent refinement of the atomic map.
Assuntos
Microscopia Crioeletrônica/normas , Gelo , Proteínas/ultraestrutura , Animais , Apoferritinas/química , Apoferritinas/ultraestrutura , Benchmarking , Microscopia Crioeletrônica/métodos , Cavalos , Gelo/normas , Proteínas/química , Tomografia/métodosRESUMO
Dynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X-ray free electron lasers have facilitated investigations of structural dynamics in proteins with unprecedented temporal and spatial resolution. Light-activated proteins are attractive targets for time-resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light-absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light absorption, rapid chemical/small-scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enables the desired chemical and/or biological output response. Time-resolved serial femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light-activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time-resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time-resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods.
Assuntos
Cristalografia por Raios X , Conformação Proteica/efeitos da radiação , Proteínas/ultraestrutura , Lasers , Luz , Modelos Moleculares , Proteínas/química , Proteínas/efeitos da radiação , Fatores de Tempo , Difração de Raios XRESUMO
Although atomic structures have been determined directly from cryo-EM density maps with high resolutions, current structure determination methods for medium resolution (5 to 10 Å) cryo-EM maps are limited by the availability of structure templates. Secondary structure traces are lines detected from a cryo-EM density map for α-helices and ß-strands of a protein. A topology of secondary structures defines the mapping between a set of sequence segments and a set of traces of secondary structures in three-dimensional space. In order to enhance accuracy in ranking secondary structure topologies, we explored a method that combines three sources of information: a set of sequence segments in 1D, a set of amino acid contact pairs in 2D, and a set of traces in 3D at the secondary structure level. A test of fourteen cases shows that the accuracy of predicted secondary structures is critical for deriving topologies. The use of significant long-range contact pairs is most effective at enriching the rank of the maximum-match topology for proteins with a large number of secondary structures, if the secondary structure prediction is fairly accurate. It was observed that the enrichment depends on the quality of initial topology candidates in this approach. We provide detailed analysis in various cases to show the potential and challenge when combining three sources of information.
Assuntos
Algoritmos , Microscopia Crioeletrônica , Bases de Dados de Proteínas , Modelos Moleculares , Proteínas/ultraestrutura , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas/químicaRESUMO
We describe new tools for the processing of electron cryo-microscopy (cryo-EM) images in the fourth major release of the RELION software. In particular, we introduce VDAM, a variable-metric gradient descent algorithm with adaptive moments estimation, for image refinement; a convolutional neural network for unsupervised selection of 2D classes; and a flexible framework for the design and execution of multiple jobs in pre-defined workflows. In addition, we present a stand-alone utility called MDCatch that links the execution of jobs within this framework with metadata gathering during microscope data acquisition. The new tools are aimed at providing fast and robust procedures for unsupervised cryo-EM structure determination, with potential applications for on-the-fly processing and the development of flexible, high-throughput structure determination pipelines. We illustrate their potential on 12 publicly available cryo-EM data sets.
Assuntos
Microscopia Crioeletrônica/métodos , Proteínas/ultraestrutura , Ribossomos/ultraestrutura , Software , AlgoritmosRESUMO
In crystallography, the phase problem can often be addressed by the careful preparation of molecular-replacement search models. This has led to the development of pipelines such as MrBUMP that can automatically identify homologous proteins from an input sequence and edit them to focus on the areas that are most conserved. Many of these approaches can be applied directly to cryo-EM to help discover, prepare and correctly place models (here called cryo-EM search models) into electrostatic potential maps. This can significantly reduce the amount of manual model building that is required for structure determination. Here, MrBUMP is repurposed to fit automatically obtained PDB-derived chains and domains into cryo-EM maps. MrBUMP was successfully able to identify and place cryo-EM search models across a range of resolutions. Methods such as map segmentation are also explored as potential routes to improved performance. Map segmentation was also found to improve the effectiveness of the pipeline for higher resolution (<8â Å) data sets.
Assuntos
Microscopia Crioeletrônica/métodos , Proteínas/ultraestrutura , Software , Animais , Bases de Dados de Proteínas , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas/químicaRESUMO
Dynein-decorated doublet microtubules (DMTs) are critical components of the oscillatory molecular machine of cilia, the axoneme, and have luminal surfaces patterned periodically by microtubule inner proteins (MIPs). Here we present an atomic model of the 48-nm repeat of a mammalian DMT, derived from a cryoelectron microscopy (cryo-EM) map of the complex isolated from bovine respiratory cilia. The structure uncovers principles of doublet microtubule organization and features specific to vertebrate cilia, including previously unknown MIPs, a luminal bundle of tektin filaments, and a pentameric dynein-docking complex. We identify a mechanism for bridging 48- to 24-nm periodicity across the microtubule wall and show that loss of the proteins involved causes defective ciliary motility and laterality abnormalities in zebrafish and mice. Our structure identifies candidate genes for diagnosis of ciliopathies and provides a framework to understand their functions in driving ciliary motility.
Assuntos
Cílios/ultraestrutura , Microscopia Crioeletrônica , Mamíferos/metabolismo , Proteínas/metabolismo , Proteínas/ultraestrutura , Sequência de Aminoácidos , Animais , Bovinos , Cílios/metabolismo , Dineínas/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Proteínas dos Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Mutação/genética , Traqueia/anatomia & histologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a 'digital twin' software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.
Assuntos
Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Proteínas/ultraestrutura , Simulação por Computador , Bases de Dados de Proteínas , Tomografia com Microscopia Eletrônica/instrumentação , SoftwareRESUMO
Ensemble-based structural modeling of flexible protein segments such as intrinsically disordered regions is a complex task often solved by selection of conformers from an initial pool based on their conformity to experimental data. However, the properties of the conformational pool are crucial, as the sampling of the conformational space should be sufficient and, in the optimal case, relatively uniform. In other words, the ideal sampling is both efficient and exhaustive. To achieve this, specialized tools are usually necessary, which might not be maintained in the long term, available on all platforms or flexible enough to be tweaked to individual needs. Here, we present an open-source and extendable pipeline to generate initial protein structure pools for use with selection-based tools to obtain ensemble models of flexible protein segments. Our method is implemented in Python and uses ChimeraX, Scwrl4, Gromacs and neighbor-dependent backbone distributions compiled and published previously by the Dunbrack lab. All these tools and data are publicly available and maintained. Our basic premise is that by using residue-specific, neighbor-dependent Ramachandran distributions, we can enhance the efficient exploration of the relevant region of the conformational space. We have also provided a straightforward way to bias the sampling towards specific conformations for selected residues by combining different conformational distributions. This allows the consideration of a priori known conformational preferences such as in the case of preformed structural elements. The open-source and modular nature of the pipeline allows easy adaptation for specific problems. We tested the pipeline on an intrinsically disordered segment of the protein Cd3ϵ and also a single-alpha helical (SAH) region by generating conformational pools and selecting ensembles matching experimental data using the CoNSEnsX+ server.
Assuntos
Biologia Computacional , Proteínas Intrinsicamente Desordenadas/ultraestrutura , Proteínas/ultraestrutura , Software/estatística & dados numéricos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Simulação de Dinâmica Molecular , Análise de Componente Principal , Conformação Proteica , Proteínas/química , Proteínas/genéticaRESUMO
Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1's active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1's conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Domínio Catalítico/genética , Humanos , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/genética , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/isolamento & purificação , Enzimas Ativadoras de Ubiquitina/ultraestrutura , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/isolamento & purificação , Enzimas de Conjugação de Ubiquitina/ultraestrutura , Difração de Raios XRESUMO
A rapid assay is described, based upon the Marangoni effect, which detects the formation of a denatured-protein film at the air-water interface (AWI) of aqueous samples. This assay requires no more than a 20 µL aliquot of sample, at a protein concentration of no more than1 mg/ml, and it can be performed with any buffer that is used to prepare grids for electron cryo-microscopy (cryo-EM). In addition, this assay provides an easy way to estimate the rate at which a given protein forms such a film at the AWI. Use of this assay is suggested as a way to pre-screen the effect of various additives and chemical modifications that one might use to optimize the preparation of grids, although the final proof of optimization still requires further screening of grids in the electron microscope. In those cases when the assay establishes that a given protein does form a sacrificial, denatured-protein monolayer, it is suggested that subsequent optimization strategies might focus on discovering how to improve the adsorption of native proteins onto that monolayer, rather than to prevent its formation. A second alternative might be to bind such proteins to the surface of rationally designed affinity grids, in order to prevent their diffusion to, and unwanted interaction with, the AWI.
Assuntos
Microscopia Crioeletrônica/métodos , Desnaturação Proteica , Proteínas/química , Proteínas/ultraestrutura , Manejo de Espécimes/métodos , Adsorção , Ar , Microscopia Crioeletrônica/instrumentação , Ferritinas/química , Ferritinas/ultraestrutura , Reprodutibilidade dos Testes , Propriedades de Superfície , Água/químicaRESUMO
In the process of drug discovery, identifying the interaction between the protein and the novel compound plays an important role. With the development of technology, deep learning methods have shown excellent performance in various situations. However, the compound-protein interaction is complicated and the features extracted by most deep models are not comprehensive, which limits the performance to a certain extent. In this paper, we proposed a multiscale convolutional network that extracted the local and global features of the protein and the topological feature of the compound using different types of convolutional networks. The results showed that our model obtained the best performance compared with the existing deep learning methods.