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1.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064029

RESUMO

The enzyme soluble guanylate cyclase (sGC) is the prototypical nitric oxide (NO) receptor in humans and other higher eukaryotes and is responsible for transducing the initial NO signal to the secondary messenger cyclic guanosine monophosphate (cGMP). Generation of cGMP in turn leads to diverse physiological effects in the cardiopulmonary, vascular, and neurological systems. Given these important downstream effects, sGC has been biochemically characterized in great detail in the four decades since its discovery. Structures of full-length sGC, however, have proven elusive until very recently. In 2019, advances in single particle cryo-electron microscopy (cryo-EM) enabled visualization of full-length sGC for the first time. This review will summarize insights revealed by the structures of sGC in the unactivated and activated states and discuss their implications in the mechanism of sGC activation.


Assuntos
Guanilil Ciclase Solúvel/metabolismo , Animais , Microscopia Crioeletrônica/métodos , GMP Cíclico/metabolismo , Humanos , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia
2.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33924132

RESUMO

Immuno-electron microscopy (Immuno-EM) is a powerful tool for identifying molecular targets with ultrastructural details in biological specimens. However, technical barriers, such as the loss of ultrastructural integrity, the decrease in antigenicity, or artifacts in the handling process, hinder the widespread use of the technique by biomedical researchers. We developed a method to overcome such challenges by combining light and electron microscopy with immunolabeling based on Tokuyasu's method. Using cryo-sectioned biological specimens, target proteins with excellent antigenicity were first immunolabeled for confocal analysis, and then the same tissue sections were further processed for electron microscopy, which provided a well-preserved ultrastructure comparable to that obtained using conventional electron microscopy. Moreover, this method does not require specifically designed correlative light and electron microscopy (CLEM) devices but rather employs conventional confocal and electron microscopes; therefore, it can be easily applied in many biomedical studies.


Assuntos
Microscopia Crioeletrônica , Secções Congeladas , Microscopia de Fluorescência , Microtomia , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Linhagem Celular , Células Cultivadas , Microscopia Crioeletrônica/instrumentação , Microscopia Crioeletrônica/métodos , Humanos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Microtomia/métodos
3.
Nat Commun ; 12(1): 2090, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33828103

RESUMO

An increasing number of density maps of biological macromolecules have been determined by cryo-electron microscopy (cryo-EM) and stored in the public database, EMDB. To interpret the structural information contained in EM density maps, alignment of maps is an essential step for structure modeling, comparison of maps, and for database search. Here, we developed VESPER, which captures the similarity of underlying molecular structures embedded in density maps by taking local gradient directions into consideration. Compared to existing methods, VESPER achieved substantially more accurate global and local alignment of maps as well as database retrieval.


Assuntos
Microscopia Crioeletrônica/métodos , Bases de Dados Factuais , Modelos Estruturais , Software , Modelos Moleculares , Conformação Proteica , Proteínas/química
4.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33926037

RESUMO

The 20S proteasome, which is composed of layered α and ß heptameric rings, is the core complex of the eukaryotic proteasome involved in proteolysis. The α7 subunit is a component of the α ring, and it self-assembles into a homo-tetradecamer consisting of two layers of α7 heptameric rings. However, the structure of the α7 double ring in solution has not been fully elucidated. We applied cryo-electron microscopy to delineate the structure of the α7 double ring in solution, revealing a structure different from the previously reported crystallographic model. The D7-symmetrical double ring was stacked with a 15° clockwise twist and a separation of 3 Å between the two rings. Two more conformations, dislocated and fully open, were also identified. Our observations suggest that the α7 double-ring structure fluctuates considerably in solution, allowing for the insertion of homologous α subunits, finally converting to the hetero-heptameric α rings in the 20S proteasome.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Microscopia Crioeletrônica/métodos , Citoplasma/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Multimerização Proteica/fisiologia , Subunidades Proteicas/metabolismo
5.
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33883267

RESUMO

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.


Assuntos
COVID-19/virologia , SARS-CoV-2/fisiologia , Replicação Viral/genética , Monofosfato de Adenosina/farmacologia , Antivirais/farmacologia , COVID-19/genética , COVID-19/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Microscopia Crioeletrônica/métodos , DNA Helicases/metabolismo , Genoma Viral , Humanos , Simulação de Dinâmica Molecular , RNA Helicases/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética
6.
J Vis Exp ; (169)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33818563

RESUMO

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , SARS-CoV-2 , Processamento de Imagem Assistida por Computador/métodos , Substâncias Macromoleculares , Reprodutibilidade dos Testes , Software
7.
Nat Commun ; 12(1): 2086, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33828102

RESUMO

Histamine receptors play important roles in various pathophysiological conditions and are effective targets for anti-allergy treatment, however the mechanism of receptor activation remain elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human H1R in complex with a Gq protein in an active conformation via a NanoBiT tethering strategy. The structure reveals that histamine activates receptor via interacting with the key residues of both transmembrane domain 3 (TM3) and TM6 to squash the binding pocket on the extracellular side and to open the cavity on the intracellular side for Gq engagement in a model of "squash to activate and expand to deactivate". The structure also reveals features for Gq coupling, including the interaction between intracellular loop 2 (ICL2) and the αN-ß junction of Gq/11 protein. The detailed analysis of our structure will provide a framework for understanding G-protein coupling selectivity and clues for designing novel antihistamines.


Assuntos
Microscopia Crioeletrônica/métodos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Histamina/química , Receptores Histamínicos/química , Sítios de Ligação , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP , Histamina/metabolismo , Humanos , Ligantes , Ligação Proteica , Domínios Proteicos , Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/metabolismo
8.
Nat Commun ; 12(1): 1620, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712624

RESUMO

Amyotrophic lateral sclerosis and several other neurodegenerative diseases are associated with brain deposits of amyloid-like aggregates formed by the C-terminal fragments of TDP-43 that contain the low complexity domain of the protein. Here, we report the cryo-EM structure of amyloid formed from the entire TDP-43 low complexity domain in vitro at pH 4. This structure reveals single protofilament fibrils containing a large (139-residue), tightly packed core. While the C-terminal part of this core region is largely planar and characterized by a small proportion of hydrophobic amino acids, the N-terminal region contains numerous hydrophobic residues and has a non-planar backbone conformation, resulting in rugged surfaces of fibril ends. The structural features found in these fibrils differ from those previously found for fibrils generated from short protein fragments. The present atomic model for TDP-43 LCD fibrils provides insight into potential structural perturbations caused by phosphorylation and disease-related mutations.


Assuntos
Amiloide/química , Microscopia Crioeletrônica/métodos , Proteínas de Ligação a DNA/química , Amiloide/genética , Amiloide/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutação Puntual , Conformação Proteica
9.
J Vis Exp ; (169)2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33779598

RESUMO

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 µm thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.


Assuntos
Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Tomografia Computadorizada por Raios X/métodos , Humanos
10.
J Vis Exp ; (169)2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33779618

RESUMO

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.


Assuntos
Microscopia Crioeletrônica/métodos , Elétrons
11.
J Vis Exp ; (168)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33720116

RESUMO

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these "time-resolved" studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.


Assuntos
Microscopia Crioeletrônica/métodos , Robótica , Congelamento , Umidade , Software , Fatores de Tempo , Interface Usuário-Computador
12.
Nat Commun ; 12(1): 1957, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785757

RESUMO

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Tomografia Computadorizada por Raios X/métodos , Algoritmos , Imageamento Tridimensional/métodos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Tamanho da Partícula , Reprodutibilidade dos Testes
13.
Nat Methods ; 18(2): 176-185, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33542510

RESUMO

Cryo-electron microscopy (cryo-EM) single-particle analysis has proven powerful in determining the structures of rigid macromolecules. However, many imaged protein complexes exhibit conformational and compositional heterogeneity that poses a major challenge to existing three-dimensional reconstruction methods. Here, we present cryoDRGN, an algorithm that leverages the representation power of deep neural networks to directly reconstruct continuous distributions of 3D density maps and map per-particle heterogeneity of single-particle cryo-EM datasets. Using cryoDRGN, we uncovered residual heterogeneity in high-resolution datasets of the 80S ribosome and the RAG complex, revealed a new structural state of the assembling 50S ribosome, and visualized large-scale continuous motions of a spliceosome complex. CryoDRGN contains interactive tools to visualize a dataset's distribution of per-particle variability, generate density maps for exploratory analysis, extract particle subsets for use with other tools and generate trajectories to visualize molecular motions. CryoDRGN is open-source software freely available at http://cryodrgn.csail.mit.edu .


Assuntos
Microscopia Crioeletrônica/métodos , Substâncias Macromoleculares/química , Redes Neurais de Computação , Estrutura Molecular
14.
Nat Methods ; 18(2): 186-193, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33542511

RESUMO

Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and inside cells. In addition to aligning individual particles, accurate registration of sample motion and three-dimensional deformation during exposures are crucial for achieving high-resolution reconstructions. Here we describe M, a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and couples a comprehensive spatial deformation model to in silico correction of electron-optical aberrations. M provides a unified optimization framework for both frame-series and tomographic tilt-series data. We show that tilt-series data can provide the same resolution as frame-series data on a purified protein specimen, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M resolves to residue level a 70S ribosome bound to an antibiotic inside intact bacterial cells. Our work provides a computational tool that facilitates structural biology in cells.


Assuntos
Antibacterianos/metabolismo , Microscopia Crioeletrônica/métodos , Ribossomos/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Interface Usuário-Computador
15.
Nat Methods ; 18(2): 156-164, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33542514

RESUMO

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.


Assuntos
Microscopia Crioeletrônica/métodos , Modelos Moleculares , Cristalografia por Raios X , Conformação Proteica , Proteínas/química
16.
Nat Commun ; 12(1): 739, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531497

RESUMO

The proteasome activator PA28αß affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28αß-iCP structure, how PA28αß regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28αß-iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28αß and iCP through XL-MS. Our structures reveal a slight leaning of PA28αß towards the α3-α4 side of iCP, disturbing the allosteric network of the gatekeeper α2/3/4 subunits, resulting in a partial open iCP gate. We find that the binding and activation mechanism of iCP by PA28αß is distinct from those of constitutive CP by the homoheptameric TbPA26 or PfPA28. Our study sheds lights on the mechanism of enzymatic activity stimulation of immunoproteasome and suggests that PA28αß-iCP has experienced profound remodeling during evolution to achieve its current level of function in immune response.


Assuntos
Microscopia Crioeletrônica/métodos , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Apresentação do Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo
17.
Proc Natl Acad Sci U S A ; 118(7)2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33526596

RESUMO

The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.


Assuntos
Amidas/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/farmacologia , Pirazinas/farmacologia , SARS-CoV-2/ultraestrutura , Amidas/química , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/química , Microscopia Crioeletrônica/métodos , Inibidores Enzimáticos/química , Pirazinas/química , Ribonucleotídeos/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Imagem Individual de Molécula/métodos
18.
Nat Chem Biol ; 17(4): 387-393, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33495647

RESUMO

Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform for transcriptome engineering and diagnostic applications. We determined the structures of Cas12g-guide RNA complexes in the absence and presence of target RNA by cryo-EM to a resolution of 3.1 Å and 4.8 Å, respectively. Cas12g adopts a bilobed structure with miniature REC2 and Nuc domains, whereas the guide RNAs fold into a flipped 'F' shape, which is primarily recognized by the REC lobe. Target RNA and the CRISPR RNA (crRNA) guide form a duplex that inserts into the central cavity between the REC and NUC lobes, inducing conformational changes in both lobes to activate Cas12g. The structural insights would facilitate the development of Cas12g-based applications.


Assuntos
Proteínas Associadas a CRISPR/ultraestrutura , RNA Guia/ultraestrutura , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Microscopia Crioeletrônica/métodos , RNA Bacteriano/química , RNA Guia/genética , Ribonucleases/genética , Ribonucleases/metabolismo , Ribonucleases/ultraestrutura
19.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33402531

RESUMO

In this paper, we present a refinement method for cryo-electron microscopy (cryo-EM) single-particle reconstruction, termed as OPUS-SSRI (Sparseness and Smoothness Regularized Imaging). In OPUS-SSRI, spatially varying sparseness and smoothness priors are incorporated to improve the regularity of electron density map, and a type of real space penalty function is designed. Moreover, we define the back-projection step as a local kernel regression and propose a first-order method to solve the resulting optimization problem. On the seven cryo-EM datasets that we tested, the average improvement in resolution by OPUS-SSRI over that from RELION 3.0, the commonly used image-processing software for single-particle cryo-EM, was 0.64 Å, with the largest improvement being 1.25 Å. We expect OPUS-SSRI to be an invaluable tool to the broad field of cryo-EM single-particle analysis. The implementation of OPUS-SSRI can be found at https://github.com/alncat/cryoem.


Assuntos
Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Imagem Individual de Molécula/métodos , Algoritmos , Biologia Computacional/métodos , Razão Sinal-Ruído , Software
20.
Biochem Biophys Res Commun ; 539: 70-76, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33422942

RESUMO

Crystallin gene mutations are responsible for about half of the congenital cataract caused by genetic disorders. L45P and Y46D mutations of γC-crystallin have been reported in patients with nuclear congenital cataract. In this study, we explored the thermal stability of wild type (WT), L45P, and Y46D mutants of γC-crystallin at low and high concentrations, as well as the effect of αA-crystallin on the thermal stability of mutants. Spectroscopic experiments were used to monitor the structural changes on temperature-gradient and time-course heating process. Intermediate morphologies were determined through cryo-electron microscopy. The thermal stability of WT and mutants at concentrations ranging up to hundreds of milligrams were assessed via the UNcle multifunctional protein stability analysis system. The results showed that L45P and Y46D mutations impaired the thermal stability of γC-crystallin at low (0.2 mg/mL) and high concentrations (up to 200 mg/mL). Notably, with increase in protein concentration, the thermal stability of L45P and Y46D mutants of γC-crystallin simultaneously decreased. Thermal stability of L45P and Y46D mutants could be rescued by αA-crystallin in a concentration-dependent manner. The dramatic decrease in thermal stability of γC-crystallin caused by L45P and Y46D mutations contributed to congenital cataract in the mature human lens.


Assuntos
Catarata/genética , Mutação , gama-Cristalinas/genética , Catarata/metabolismo , Catarata/patologia , Microscopia Crioeletrônica/métodos , Humanos , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura , gama-Cristalinas/química , gama-Cristalinas/metabolismo
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