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1.
Science ; 373(6557): 871-876, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34282049

RESUMO

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.


Assuntos
Aprendizado Profundo , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas ADAM/química , Sequência de Aminoácidos , Simulação por Computador , Microscopia Crioeletrônica , Cristalografia por Raios X , Bases de Dados de Proteínas , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos/química , Redes Neurais de Computação , Subunidades Proteicas/química , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/química , Esfingosina N-Aciltransferase/química
2.
Curr Protoc Protein Sci ; 102(1): e116, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33320432

RESUMO

While native proteins cover diverse structural spaces and achieve various biological events, not many of them can directly serve human needs. One reason is that the native proteins usually contain idiosyncrasies evolved for their native functions but disfavoring engineering requirements. To overcome this issue, one strategy is to create de novo proteins which are designed to possess improved stability, high environmental tolerance, and enhanced engineering potential. Compared to other protein engineering strategies, in silico design of de novo proteins has significantly expanded the protein structural and sequence spaces, reduced wet lab workload, and incorporated engineered features in a guided and efficient manner. In the Baker laboratory we have been applying a design pipeline that uses the blueprint builder to design different folds of de novo proteins, and have successfully obtained libraries of de novo proteins with improved stability and engineering potential. In this article, we will use the design of de novo ß-barrel proteins as an example to describe the principles and basic procedures of the blueprint builder-based design pipeline. © 2020 Wiley Periodicals LLC. Basic Protocol 1: The construction of blueprints Alternate Protocol: Build blueprints based on existing protein .pdb files Basic Protocol 2: De novo protein design pipeline using the blueprint builder.


Assuntos
Simulação por Computador , Conformação Proteica , Engenharia de Proteínas , Humanos
3.
Proteins ; 87(12): 1276-1282, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31325340

RESUMO

Because proteins generally fold to their lowest free energy states, energy-guided refinement in principle should be able to systematically improve the quality of protein structure models generated using homologous structure or co-evolution derived information. However, because of the high dimensionality of the search space, there are far more ways to degrade the quality of a near native model than to improve it, and hence, refinement methods are very sensitive to energy function errors. In the 13th Critial Assessment of techniques for protein Structure Prediction (CASP13), we sought to carry out a thorough search for low energy states in the neighborhood of a starting model using restraints to avoid straying too far. The approach was reasonably successful in improving both regions largely incorrect in the starting models as well as core regions that started out closer to the correct structure. Models with GDT-HA over 70 were obtained for five targets and for one of those, an accuracy of 0.5 å backbone root-mean-square deviation (RMSD) was achieved. An important current challenge is to improve performance in refining oligomers and larger proteins, for which the search problem remains extremely difficult.


Assuntos
Biologia Computacional/métodos , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Algoritmos , Modelos Moleculares , Reprodutibilidade dos Testes , Termodinâmica
4.
Nucleic Acids Res ; 47(W1): W451-W455, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31001635

RESUMO

The 3D structure of a protein can be predicted from its amino acid sequence with high accuracy for a large fraction of cases because of the availability of large quantities of experimental data and the advance of computational algorithms. Recently, deep learning methods exploiting the coevolution information obtained by comparing related protein sequences have been successfully used to generate highly accurate model structures even in the absence of template structure information. However, structures predicted based on either template structures or related sequences require further improvement in regions for which information is missing. Refining a predicted protein structure with insufficient information on certain regions is critical because these regions may be connected to functional specificity that is not conserved among related proteins. The GalaxyRefine2 web server, freely available via http://galaxy.seoklab.org/refine2, is an upgraded version of the GalaxyRefine protein structure refinement server and reflects recent developments successfully tested through CASP blind prediction experiments. This method adopts an iterative optimization approach involving various structure move sets to refine both local and global structures. The estimation of local error and hybridization of available homolog structures are also employed for effective conformation search.


Assuntos
Conformação Proteica , Software , Modelos Moleculares , Análise de Sequência de Proteína
5.
Proc Natl Acad Sci U S A ; 115(35): 8787-8792, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30104375

RESUMO

Wnt signaling is initiated by Wnt ligand binding to the extracellular ligand binding domain, called the cysteine-rich domain (CRD), of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specifically interacts with Fzd4 to activate ß-catenin-dependent canonical Wnt signaling. Much of the molecular basis that confers Norrin selectivity in binding to Fzd4 was revealed through the structural study of the Fzd4CRD-Norrin complex. However, how the ligand interaction, seemingly localized at the CRD, is transmitted across full-length Fzd4 to the cytoplasm remains largely unknown. Here, we show that a flexible linker domain, which connects the CRD to the transmembrane domain, plays an important role in Norrin signaling. The linker domain directly contributes to the high-affinity interaction between Fzd4 and Norrin as shown by ∼10-fold higher binding affinity of Fzd4CRD to Norrin in the presence of the linker. Swapping the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin signaling, suggesting the importance of the linker in ligand-specific cellular response. In addition, structural dynamics of Fzd4 associated with Norrin binding investigated by hydrogen/deuterium exchange MS revealed Norrin-induced conformational changes on the linker domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A mutations at ICL3, and C-terminal tail truncation displayed reduced ß-catenin-dependent signaling activity, indicating the functional significance of these sites. Together, our results provide functional and biochemical dissection of Fzd4 in Norrin signaling.


Assuntos
Proteínas do Olho/química , Receptores Frizzled/química , Proteínas do Tecido Nervoso/química , Via de Sinalização Wnt , Animais , Proteínas do Olho/metabolismo , Receptores Frizzled/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
6.
Sci Rep ; 8(1): 9939, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29967418

RESUMO

Every two years groups worldwide participate in the Critical Assessment of Protein Structure Prediction (CASP) experiment to blindly test the strengths and weaknesses of their computational methods. CASP has significantly advanced the field but many hurdles still remain, which may require new ideas and collaborations. In 2012 a web-based effort called WeFold, was initiated to promote collaboration within the CASP community and attract researchers from other fields to contribute new ideas to CASP. Members of the WeFold coopetition (cooperation and competition) participated in CASP as individual teams, but also shared components of their methods to create hybrid pipelines and actively contributed to this effort. We assert that the scale and diversity of integrative prediction pipelines could not have been achieved by any individual lab or even by any collaboration among a few partners. The models contributed by the participating groups and generated by the pipelines are publicly available at the WeFold website providing a wealth of data that remains to be tapped. Here, we analyze the results of the 2014 and 2016 pipelines showing improvements according to the CASP assessment as well as areas that require further adjustments and research.


Assuntos
Caspase 12/metabolismo , Caspases/metabolismo , Biologia Computacional/métodos , Modelos Moleculares , Software , Caspase 12/química , Caspases/química , Humanos , Conformação Proteica
7.
J Chem Inf Model ; 58(6): 1234-1243, 2018 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-29786430

RESUMO

The second extracellular loops (ECL2s) of G-protein-coupled receptors (GPCRs) are often involved in GPCR functions, and their structures have important implications in drug discovery. However, structure prediction of ECL2 is difficult because of its long length and the structural diversity among different GPCRs. In this study, a new ECL2 conformational sampling method involving both template-based and ab initio sampling was developed. Inspired by the observation of similar ECL2 structures of closely related GPCRs, a template-based sampling method employing loop structure templates selected from the structure database was developed. A new metric for evaluating similarity of the target loop to templates was introduced for template selection. An ab initio loop sampling method was also developed to treat cases without highly similar templates. The ab initio method is based on the previously developed fragment assembly and loop closure method. A new sampling component that takes advantage of secondary structure prediction was added. In addition, a conserved disulfide bridge restraining ECL2 conformation was predicted and analytically incorporated into sampling, reducing the effective dimension of the conformational search space. The sampling method was combined with an existing energy function for comparison with previously reported loop structure prediction methods, and the benchmark test demonstrated outstanding performance.


Assuntos
Receptores Acoplados a Proteínas G/química , Animais , Bases de Dados de Proteínas , Dissulfetos/química , Humanos , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína
8.
Proteins ; 86 Suppl 1: 168-176, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29044810

RESUMO

Advances in protein model refinement techniques are required as diverse sources of protein structure information are available from low-resolution experiments or informatics-based computations such as cryo-EM, NMR, homology models, or predicted residue contacts. Given semi-reliable or incomplete structural information, structure quality of a protein model has to be improved by ab initio methods such as energy-based simulation. In this study, we describe a new automatic refinement server method designed to improve locally inaccurate regions and overall structure simultaneously. Locally inaccurate regions may occur in protein structures due to non-convergent or missing information in template structures used in homology modeling or due to intrinsic structural flexibilities not resolved by experimental techniques. However, such variable or dynamic regions often play important functional roles by participating in interactions with other biomolecules or in transitions between different functional states. The new refinement method introduced here utilizes diverse types of geometric operators which drive both local and global changes, and the effect of structure changes and relaxations are accumulated. This resulted in consistent refinement of both local and global structural features. Performance of this method in CASP12 is discussed.


Assuntos
Biologia Computacional/métodos , Aprendizado de Máquina , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Algoritmos , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Análise de Sequência de Proteína
9.
J Biol Chem ; 292(40): 16477-16490, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28842483

RESUMO

Stable tissue integrity during embryonic development relies on the function of the cadherin·catenin complex (CCC). The Caenorhabditis elegans CCC is a useful paradigm for analyzing in vivo requirements for specific interactions among the core components of the CCC, and it provides a unique opportunity to examine evolutionarily conserved mechanisms that govern the interaction between α- and ß-catenin. HMP-1, unlike its mammalian homolog α-catenin, is constitutively monomeric, and its binding affinity for HMP-2/ß-catenin is higher than that of α-catenin for ß-catenin. A crystal structure shows that the HMP-1·HMP-2 complex forms a five-helical bundle structure distinct from the structure of the mammalian α-catenin·ß-catenin complex. Deletion analysis based on the crystal structure shows that the first helix of HMP-1 is necessary for binding HMP-2 avidly in vitro and for efficient recruitment of HMP-1 to adherens junctions in embryos. HMP-2 Ser-47 and Tyr-69 flank its binding interface with HMP-1, and we show that phosphomimetic mutations at these two sites decrease binding affinity of HMP-1 to HMP-2 by 40-100-fold in vitro. In vivo experiments using HMP-2 S47E and Y69E mutants showed that they are unable to rescue hmp-2(zu364) mutants, suggesting that phosphorylation of HMP-2 on Ser-47 and Tyr-69 could be important for regulating CCC formation in C. elegans Our data provide novel insights into how cadherin-dependent cell-cell adhesion is modulated in metazoans by conserved elements as well as features unique to specific organisms.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Comunicação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Complexos Multiproteicos/metabolismo , alfa Catenina/metabolismo , Substituição de Aminoácidos , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Adesão Celular/fisiologia , Cristalografia por Raios X , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação de Sentido Incorreto , Estrutura Quaternária de Proteína , alfa Catenina/química , alfa Catenina/genética
10.
Hum Mutat ; 38(9): 1123-1131, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28370845

RESUMO

The Critical Assessment of Genome Interpretation (CAGI) is a global community experiment to objectively assess computational methods for predicting phenotypic impacts of genomic variation. One of the 2015-2016 competitions focused on predicting the influence of mutations on the allosteric regulation of human liver pyruvate kinase. More than 30 different researchers accessed the challenge data. However, only four groups accepted the challenge. Features used for predictions ranged from evolutionary constraints, mutant site locations relative to active and effector binding sites, and computational docking outputs. Despite the range of expertise and strategies used by predictors, the best predictions were marginally greater than random for modified allostery resulting from mutations. In contrast, several groups successfully predicted which mutations severely reduced enzymatic activity. Nonetheless, poor predictions of allostery stands in stark contrast to the impression left by more than 700 PubMed entries identified using the identifiers "computational + allosteric." This contrast highlights a specialized need for new computational tools and utilization of benchmarks that focus on allosteric regulation.


Assuntos
Benchmarking/métodos , Piruvato Quinase/química , Piruvato Quinase/genética , Regulação Alostérica , Sítio Alostérico , Biologia Computacional/métodos , Bases de Dados Genéticas , Frutosedifosfatos/metabolismo , Humanos , Modelos Moleculares , Mutação , Piruvato Quinase/metabolismo
11.
Proteins ; 85(3): 399-407, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27770545

RESUMO

Many proteins function as homo- or hetero-oligomers; therefore, attempts to understand and regulate protein functions require knowledge of protein oligomer structures. The number of available experimental protein structures is increasing, and oligomer structures can be predicted using the experimental structures of related proteins as templates. However, template-based models may have errors due to sequence differences between the target and template proteins, which can lead to functional differences. Such structural differences may be predicted by loop modeling of local regions or refinement of the overall structure. In CAPRI (Critical Assessment of PRotein Interactions) round 30, we used recently developed features of the GALAXY protein modeling package, including template-based structure prediction, loop modeling, model refinement, and protein-protein docking to predict protein complex structures from amino acid sequences. Out of the 25 CAPRI targets, medium and acceptable quality models were obtained for 14 and 1 target(s), respectively, for which proper oligomer or monomer templates could be detected. Symmetric interface loop modeling on oligomer model structures successfully improved model quality, while loop modeling on monomer model structures failed. Overall refinement of the predicted oligomer structures consistently improved the model quality, in particular in interface contacts. Proteins 2017; 85:399-407. © 2016 Wiley Periodicals, Inc.


Assuntos
Algoritmos , Biologia Computacional/métodos , Simulação de Acoplamento Molecular/métodos , Proteínas/química , Sequência de Aminoácidos , Benchmarking , Sítios de Ligação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Projetos de Pesquisa , Software , Homologia Estrutural de Proteína
12.
Nucleic Acids Res ; 44(W1): W502-6, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27131365

RESUMO

G-protein-coupled receptors (GPCRs) play important physiological roles related to signal transduction and form a major group of drug targets. Prediction of GPCR-ligand complex structures has therefore important implications to drug discovery. With previously available servers, it was only possible to first predict GPCR structures by homology modeling and then perform ligand docking on the model structures. However, model structures generated without explicit consideration of specific ligands of interest can be inaccurate because GPCR structures can be affected by ligand binding. The Galaxy7TM server, freely accessible at http://galaxy.seoklab.org/7TM, improves an input GPCR structure by simultaneous ligand docking and flexible structure refinement using GALAXY methods. The server shows better performance in both ligand docking and GPCR structure refinement than commonly used programs AutoDock Vina and Rosetta MPrelax, respectively.


Assuntos
Internet , Simulação de Acoplamento Molecular , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Software , Azepinas/química , Azepinas/metabolismo , Humanos , Ligantes , Receptores de Orexina/química , Receptores de Orexina/metabolismo , Triazóis/química , Triazóis/metabolismo
13.
Proteins ; 84 Suppl 1: 293-301, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-26172288

RESUMO

Protein structures predicted by state-of-the-art template-based methods may still have errors when the template proteins are not similar enough to the target protein. Overall target structure may deviate from the template structures owing to differences in sequences. Structural information for some local regions such as loops may not be available when there are sequence insertions or deletions. Those structural aspects that originate from deviations from templates can be dealt with by ab initio structure refinement methods to further improve model accuracy. In the CASP11 refinement experiment, we tested three different refinement methods that utilize overall structure relaxation, loop modeling, and quality assessment of multiple initial structures. From this experiment, we conclude that the overall relaxation method can consistently improve model quality. Loop modeling is the most useful when the initial model structure is high quality, with GDT-HA >60. The method that used multiple initial structures further refined the already refined models; the minor improvements with this method raise the issue of problem with the current energy function. Future research directions are also discussed. Proteins 2016; 84(Suppl 1):293-301. © 2015 Wiley Periodicals, Inc.


Assuntos
Biologia Computacional/estatística & dados numéricos , Modelos Moleculares , Modelos Estatísticos , Proteínas/química , Software , Algoritmos , Motivos de Aminoácidos , Biologia Computacional/métodos , Simulação por Computador , Humanos , Internet , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Termodinâmica
14.
J Chem Inf Model ; 56(6): 988-95, 2016 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-26583962

RESUMO

We analyze the results of the GalaxyDock protein-ligand docking program in the two recent experiments of Community Structure-Activity Resource (CSAR), CSAR 2013 and 2014. GalaxyDock performs global optimization of a modified AutoDock3 energy function by employing the conformational space annealing method. The energy function of GalaxyDock is quite sensitive to atomic clashes. Such energy functions can be effective for sampling physically correct conformations but may not be effective for scoring when conformations are not fully optimized. In phase 1 of CSAR 2013, we successfully selected all four true binders of digoxigenin along with three false positives. However, the energy values were rather high due to insufficient optimization of the conformations docked to homology models. A posteriori relaxation of the model complex structures by GalaxyRefine improved the docking energy values and differentiated the true binders from the false positives better. In the scoring test of CSAR 2013 phase 2, we selected the best poses for each of the two targets. The results of CSAR 2013 phase 3 suggested that an improved method for generating initial conformations for GalaxyDock is necessary for targets involving bulky ligands. Finally, combining existing binding information with GalaxyDock energy-based optimization may be needed for more accurate binding affinity prediction.


Assuntos
Simulação de Acoplamento Molecular , Benchmarking , Ligantes , Conformação Proteica , Proteínas/química , Proteínas/metabolismo , Relação Estrutura-Atividade
15.
PLoS One ; 9(11): e113811, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25419655

RESUMO

Protein loop modeling is a tool for predicting protein local structures of particular interest, providing opportunities for applications involving protein structure prediction and de novo protein design. Until recently, the majority of loop modeling methods have been developed and tested by reconstructing loops in frameworks of experimentally resolved structures. In many practical applications, however, the protein loops to be modeled are located in inaccurate structural environments. These include loops in model structures, low-resolution experimental structures, or experimental structures of different functional forms. Accordingly, discrepancies in the accuracy of the structural environment assumed in development of the method and that in practical applications present additional challenges to modern loop modeling methods. This study demonstrates a new strategy for employing a hybrid energy function combining physics-based and knowledge-based components to help tackle this challenge. The hybrid energy function is designed to combine the strengths of each energy component, simultaneously maintaining accurate loop structure prediction in a high-resolution framework structure and tolerating minor environmental errors in low-resolution structures. A loop modeling method based on global optimization of this new energy function is tested on loop targets situated in different levels of environmental errors, ranging from experimental structures to structures perturbed in backbone as well as side chains and template-based model structures. The new method performs comparably to force field-based approaches in loop reconstruction in crystal structures and better in loop prediction in inaccurate framework structures. This result suggests that higher-accuracy predictions would be possible for a broader range of applications. The web server for this method is available at http://galaxy.seoklab.org/loop with the PS2 option for the scoring function.


Assuntos
Biologia Computacional/métodos , Modelos Moleculares , Conformação Proteica , Proteínas/química , Algoritmos , Cristalografia por Raios X , Cinética , Reprodutibilidade dos Testes , Termodinâmica
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