Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.855
Filtrar
2.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 332-339, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254057

RESUMO

Confidence maps provide complementary information for interpreting cryo-EM densities as they indicate statistical significance with respect to background noise. They can be thresholded by specifying the expected false-discovery rate (FDR), and the displayed volume shows the parts of the map that have the corresponding level of significance. Here, the basic statistical concepts of confidence maps are reviewed and practical guidance is provided for their interpretation and usage inside the CCP-EM suite. Limitations of the approach are discussed and extensions towards other error criteria such as the family-wise error rate are presented. The observed map features can be rendered at a common isosurface threshold, which is particularly beneficial for the interpretation of weak and noisy densities. In the current article, a practical guide is provided to the recommended usage of confidence maps.


Assuntos
Microscopia Crioeletrônica/métodos , ATPases Bacterianas Próton-Translocadoras/química , Gráficos por Computador , Modelos Moleculares , Conformação Proteica , Ribossomos/química , Eletricidade Estática , Estatística como Assunto , Vírus do Mosaico do Tabaco , Interface Usuário-Computador
3.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 340-349, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254058

RESUMO

Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air-water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.


Assuntos
Microscopia Crioeletrônica/métodos , Manejo de Espécimes/métodos
4.
Nat Commun ; 11(1): 1772, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286308

RESUMO

Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization.


Assuntos
Microscopia Crioeletrônica/métodos , Espectrometria de Massas/métodos , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/ultraestrutura , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica
5.
Proc Natl Acad Sci U S A ; 117(11): 5895-5906, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123115

RESUMO

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key ß-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica/métodos , Mycobacterium tuberculosis/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Cristalografia por Raios X , Endopeptidase Clp/química , Endopeptidase Clp/metabolismo , Escherichia coli , Homeostase , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise
6.
Proc Natl Acad Sci U S A ; 117(12): 6866-6874, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32161130

RESUMO

Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer's disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.


Assuntos
Amiloide/genética , Amiloide/metabolismo , Encéfalo/metabolismo , Microscopia Crioeletrônica/métodos , Ouro/química , Nanopartículas Metálicas/química , Polimorfismo Genético , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/química , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Emaranhados Neurofibrilares
7.
Proc Natl Acad Sci U S A ; 117(12): 6784-6791, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152109

RESUMO

Infection by Rhinovirus-C (RV-C), a species of Picornaviridae Enterovirus, is strongly associated with childhood asthma exacerbations. Cellular binding and entry by all RV-C, which trigger these episodes, is mediated by the first extracellular domain (EC1) of cadherin-related protein 3 (CDHR3), a surface cadherin-like protein expressed primarily on the apical surfaces of ciliated airway epithelial cells. Although recombinant EC1 is a potent inhibitor of viral infection, there is no molecular description of this protein or its binding site on RV-C. Here we present cryo-electron microscopy (EM) data resolving the EC1 and EC1+2 domains of human CDHR3 complexed with viral isolate C15a. Structure-suggested residues contributing to required interfaces on both EC1 and C15a were probed and identified by mutagenesis studies with four different RV-C genotypes. In contrast to most other rhinoviruses, which bind intercellular adhesion molecule 1 receptors via a capsid protein VP1-specific fivefold canyon feature, the CDHR3 EC1 contacts C15a, and presumably all RV-Cs, in a unique cohesive footprint near the threefold vertex, encompassing residues primarily from viral protein VP3, but also from VP1 and VP2. The EC1+2 footprint on C15a is similar to that of EC1 alone but shows that steric hindrance imposed by EC2 would likely prevent multiprotein binding by the native receptor at any singular threefold vertex. Definition of the molecular interface between the RV-Cs and their receptors provides new avenues that can be explored for potential antiviral therapies.


Assuntos
Caderinas/química , Caderinas/metabolismo , Microscopia Crioeletrônica/métodos , Enterovirus/química , Enterovirus/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Virais/metabolismo , Enterovirus/classificação , Infecções por Enterovirus/virologia , Células HeLa , Humanos , Modelos Moleculares , Conformação Proteica
8.
Nat Commun ; 11(1): 1598, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221310

RESUMO

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.


Assuntos
Anticorpos/farmacologia , Microscopia Crioeletrônica/métodos , Proteínas de Membrana/química , Anticorpos/química , Membrana Celular/metabolismo , Técnicas de Visualização da Superfície Celular , Cristalografia por Raios X , Humanos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Polímeros , Propilaminas , Ligação Proteica , Conformação Proteica
9.
PLoS One ; 15(3): e0230198, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32155221

RESUMO

Hsp104 is a hexameric AAA+ yeast disaggregase capable of solubilizing disordered aggregates and amyloid. Hsp104 couples ATP hydrolysis to polypeptide translocation through its central channel. Substrate binding by Hsp104 is mediated primarily by two conserved tyrosine residues in nucleotide binding domain (NBD) 1 and NBD2. Recent structural studies have revealed that an additional tyrosine residue (Y650) located in NBD2 appears to contact substrate and may play an important role in Hsp104 function. Here, we functionally analyze the properties of this proposed Hsp104 -substrate interaction. We find that Y650 is not essential for Hsp104 to confer thermotolerance. Supporting these findings, in a potentiated Hsp104 variant background, the Y650A mutation does not abolish potentiation. However, modulation of this site does have subtle effects on the activity of this potentiated Hsp104 variant. We therefore suggest that while Y650 is not essential for Hsp104 function, its modulation may be useful for fine-tuning Hsp104 properties.


Assuntos
Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica/métodos , Proteínas de Choque Térmico HSP70/metabolismo , Modelos Moleculares , Ligação Proteica/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
10.
Nat Commun ; 11(1): 1506, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198400

RESUMO

Sorting nexins (SNX) are a family of PX domain-containing proteins with pivotal roles in trafficking and signaling. SNX-BARs, which also have a curvature-generating Bin/Amphiphysin/Rvs (BAR) domain, have membrane-remodeling functions, particularly at the endosome. The minimal PX-BAR module is a dimer mediated by BAR-BAR interactions. Many SNX-BAR proteins, however, additionally have low-complexity N-terminal regions of unknown function. Here, we present the cryo-EM structure of the full-length SNX-BAR Mvp1, which is an autoinhibited tetramer. The tetramer is a dimer of dimers, wherein the membrane-interacting BAR surfaces are sequestered and the PX lipid-binding sites are occluded. The N-terminal low-complexity region of Mvp1 is essential for tetramerization. Mvp1 lacking its N-terminus is dimeric and exhibits enhanced membrane association. Membrane binding and remodeling by Mvp1 therefore requires unmasking of the PX and BAR domain lipid-interacting surfaces. This work reveals a tetrameric configuration of a SNX-BAR protein that provides critical insight into SNX-BAR function and regulation.


Assuntos
Microscopia Crioeletrônica/métodos , Prolapso da Valva Mitral/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nexinas de Classificação/metabolismo , Sítios de Ligação , Biofísica , Membrana Celular/metabolismo , Endossomos/metabolismo , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Domínios Proteicos/genética , Transporte Proteico , Saccharomyces cerevisiae/genética
11.
Nat Commun ; 11(1): 876, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054835

RESUMO

Cryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution in principle can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available and might be used to further develop processing routines.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Benchmarking , Microscopia Crioeletrônica/normas , Bases de Dados Factuais , Tomografia com Microscopia Eletrônica/normas , HIV-1/química , HIV-1/ultraestrutura , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Biologia Molecular/métodos , Biologia Molecular/normas , Vírion/química , Vírion/ultraestrutura
12.
Science ; 367(6479): 806-810, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32001525

RESUMO

Although second-generation HIV integrase strand-transfer inhibitors (INSTIs) are prescribed throughout the world, the mechanistic basis for the superiority of these drugs is poorly understood. We used single-particle cryo-electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near-atomic resolution. Glutamine-148→histidine (Q148H) and glycine-140→serine (G140S) amino acid substitutions in integrase that result in clinical INSTI failure perturb optimal magnesium ion coordination in the enzyme active site. The expanded chemical scaffolds of second-generation compounds mediate interactions with the protein backbone that are critical for antagonizing viruses containing the Q148H and G140S mutations. Our results reveal that binding to magnesium ions underpins a fundamental weakness of the INSTI pharmacophore that is exploited by the virus to engender resistance and provide a structural framework for the development of this class of anti-HIV/AIDS therapeutics.


Assuntos
Farmacorresistência Viral , Inibidores de Integrase de HIV/química , Integrase de HIV/química , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Substituição de Aminoácidos/genética , Domínio Catalítico , Microscopia Crioeletrônica/métodos , Glutamina/genética , Glicina/genética , Integrase de HIV/genética , Inibidores de Integrase de HIV/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Histidina/genética , Humanos , Magnésio/química , Mutação , Serina/genética , Imagem Individual de Molécula/métodos
13.
PLoS Pathog ; 16(2): e1008314, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32069326

RESUMO

Staphylococcus aureus is a common cause of infections in humans. The emergence of virulent, antibiotic-resistant strains of S. aureus is a significant public health concern. Most virulence and resistance factors in S. aureus are encoded by mobile genetic elements, and transduction by bacteriophages represents the main mechanism for horizontal gene transfer. The baseplate is a specialized structure at the tip of bacteriophage tails that plays key roles in host recognition, cell wall penetration, and DNA ejection. We have used high-resolution cryo-electron microscopy to determine the structure of the S. aureus bacteriophage 80α baseplate at 3.75 Å resolution, allowing atomic models to be built for most of the major tail and baseplate proteins, including two tail fibers, the receptor binding protein, and part of the tape measure protein. Our structure provides a structural basis for understanding host recognition, cell wall penetration and DNA ejection in viruses infecting Gram-positive bacteria. Comparison to other phages demonstrates the modular design of baseplate proteins, and the adaptations to the host that take place during the evolution of staphylococci and other pathogens.


Assuntos
Bacteriófagos/metabolismo , Bacteriófagos/ultraestrutura , Staphylococcus aureus/genética , Bacteriófagos/genética , Microscopia Crioeletrônica/métodos , Transferência Genética Horizontal/genética , Humanos , Modelos Moleculares , Ligação Proteica/genética , Conformação Proteica , Staphylococcus aureus/ultraestrutura , Staphylococcus aureus/virologia , Vírion/genética
14.
Nat Chem Biol ; 16(3): 231-239, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32080621

RESUMO

Although viruses are extremely diverse in shape and size, evolution has led to a limited number of viral classes or lineages, which is probably linked to the assembly constraints of a viable capsid. Viral assembly mechanisms are restricted to two general pathways, (i) co-assembly of capsid proteins and single-stranded nucleic acids and (ii) a sequential mechanism in which scaffolding-mediated capsid precursor assembly is followed by genome packaging. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), which are revolutionizing structural biology, are central to determining the high-resolution structures of many viral assemblies as well as those of assembly intermediates. This wealth of cryo-EM data has also led to the development and redesign of virus-based platforms for biomedical and biotechnological applications. In this Review, we will discuss recent viral assembly analyses by cryo-EM and cryo-ET showing how natural assembly mechanisms are used to encapsulate heterologous cargos including chemicals, enzymes, and/or nucleic acids for a variety of nanotechnological applications.


Assuntos
Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Montagem de Vírus/fisiologia , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/fisiologia , Cristalografia por Raios X , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica
16.
Mol Cell ; 77(3): 656-668.e5, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004469

RESUMO

Class B G protein-coupled receptors (GPCRs) are important therapeutic targets for major diseases. Here, we present structures of peptide and Gs-bound pituitary adenylate cyclase-activating peptide, PAC1 receptor, and corticotropin-releasing factor (CRF), (CRF1) receptor. Together with recently solved structures, these provide coverage of the major class B GPCR subfamilies. Diverse orientations of the extracellular domain to the receptor core in different receptors are at least partially dependent on evolutionary conservation in the structure and nature of peptide interactions. Differences in peptide interactions to the receptor core also influence the interlinked TM2-TM1-TM6/ECL3/TM7 domain, and this is likely important in their diverse signaling. However, common conformational reorganization of ECL2, linked to reorganization of ICL2, modulates G protein contacts. Comparison between receptors reveals ICL2 as a key domain forming dynamic G protein interactions in a receptor- and ligand-specific manner. This work advances our understanding of class B GPCR activation and Gs coupling.


Assuntos
Receptores de Hormônio Liberador da Corticotropina/ultraestrutura , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/ultraestrutura , Sequência de Aminoácidos , Microscopia Crioeletrônica/métodos , Encefalinas , Humanos , Ligantes , Modelos Moleculares , Peptídeos , Precursores de Proteínas , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Acoplados a Proteínas-G/ultraestrutura , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Transdução de Sinais
17.
Mol Cell ; 77(3): 669-680.e4, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004470

RESUMO

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.


Assuntos
Receptores de Hormônio Liberador da Corticotropina/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Hormônio Liberador da Corticotropina , Microscopia Crioeletrônica/métodos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Peptídeos/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Urocortinas/metabolismo
18.
Acta Crystallogr D Struct Biol ; 76(Pt 2): 94-101, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32038040

RESUMO

Helical reconstruction in RELION is increasingly being used to determine the atomic structures of amyloid filaments from electron cryo-microscopy (cryo-EM) images. However, because the energy landscape of amyloid refinements is typically fraught with local optima, amyloid structure determination is often difficult. This paper aims to help RELION users in this process. It discusses aspects of helical reconstruction that are particularly relevant to amyloids, it illustrates the problem of local optima in refinement and how to detect them, and it introduces a new method to calculate 3D initial models from reference-free 2D class averages. By providing starting models that are closer to the global optimum, this method makes amyloid structure determination easier. All methods described are open-source and distributed within RELION-3.1. Their use is illustrated using a publicly available data set on tau filaments from the brain of an individual with Alzheimer's disease.


Assuntos
Amiloide/química , Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Algoritmos , Doença de Alzheimer , Amiloide/ultraestrutura , Humanos , Software , Proteínas tau/química
19.
Nat Commun ; 11(1): 541, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992713

RESUMO

The fast development of high-resolution electron microscopy (EM) demands a background-noise-free substrate to support the specimens, where atomically thin graphene membranes can serve as an ideal candidate. Yet the preparation of robust and ultraclean graphene EM grids remains challenging. Here we present a polymer- and transfer-free direct-etching method for batch fabrication of robust ultraclean graphene grids through membrane tension modulation. Loading samples on such graphene grids enables the detection of single metal atoms and atomic-resolution imaging of the iron core of ferritin molecules at both room- and cryo-temperature. The same kind of hydrophilic graphene grid allows the formation of ultrathin vitrified ice layer embedded most protein particles at the graphene-water interface, which facilitates cryo-EM 3D reconstruction of archaea 20S proteasomes at a record high resolution of ~2.36 Å. Our results demonstrate the significant improvements in image quality using the graphene grids and expand the scope of EM imaging.


Assuntos
Grafite/química , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Fenômenos Químicos , Microscopia Crioeletrônica/métodos , Elétrons , Desenho de Equipamento , Processamento de Imagem Assistida por Computador , Membranas , Polímeros , Proteínas
20.
Nat Methods ; 17(2): 209-216, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907446

RESUMO

With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membrane-bound complexes and an unsupervised classification (PySeg). Applications to intact cells and isolated endoplasmic reticulum (ER) allowed us to detect and classify small protein complexes. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Spatial distribution analysis showed that ER complexes have different localization patterns forming nanodomains. Therefore, this procedure allows a comprehensive detection and structural analysis of complexes in situ.


Assuntos
Microscopia Crioeletrônica/métodos , Animais , Análise por Conglomerados , Masculino , Camundongos , Ratos , Ratos Wistar , Reprodutibilidade dos Testes
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA