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1.
Nat Commun ; 11(1): 4768, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958747

RESUMO

Detection and identification of proteins are typically achieved by analyzing protein size, charge, mobility and binding to antibodies, which are critical for biomedical research and disease diagnosis and treatment. Despite the importance, measuring these quantities with one technology and at the single-molecule level has not been possible. Here we tether a protein to a surface with a flexible polymer, drive it into oscillation with an electric field, and image the oscillation with a near field optical imaging method, from which we determine the size, charge, and mobility of the protein. We also measure antibody binding and conformation changes in the protein. The work demonstrates a capability for comprehensive protein analysis and precision protein biomarker detection at the single molecule level.


Assuntos
Proteínas/química , Proteínas/metabolismo , Imagem Individual de Molécula/métodos , Análise de Fourier , Ligantes , Polietilenoglicóis/química , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Compostos de Estanho/química
2.
Nat Commun ; 11(1): 4259, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848156

RESUMO

The plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available to manipulate membrane protein location at the single-molecule level. Here, we use fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to control their movement. FMNPs allow single-particle tracking (SPT) at 10 nm and 5 ms spatiotemporal resolution, and using a magnetic needle, we pull membrane components laterally with femtonewton-range forces. In this way, we drag membrane proteins over the surface of living cells. Doing so, we detect barriers which we could localize to the submembrane actin cytoskeleton by super-resolution microscopy. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion.


Assuntos
Nanopartículas de Magnetita , Proteínas de Membrana/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Proteínas de Fluorescência Verde/metabolismo , Campos Magnéticos , Lipídeos de Membrana/metabolismo , Microscopia de Fluorescência , Nanotecnologia , Imagem Individual de Molécula/métodos
3.
Nat Commun ; 11(1): 4281, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855416

RESUMO

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.


Assuntos
Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Iniciação da Transcrição Genética , Trifosfato de Adenosina , DNA Fúngico/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Nat Commun ; 11(1): 3781, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728047

RESUMO

Nanomechanical mass spectrometry has proven to be well suited for the analysis of high mass species such as viruses. Still, the use of one-dimensional devices such as vibrating beams forces a trade-off between analysis time and mass resolution. Complex readout schemes are also required to simultaneously monitor multiple resonance modes, which degrades resolution. These issues restrict nanomechanical MS to specific species. We demonstrate here single-particle mass spectrometry with nano-optomechanical resonators fabricated with a Very Large Scale Integration process. The unique motion sensitivity of optomechanics allows designs that are impervious to particle position, stiffness or shape, opening the way to the analysis of large aspect ratio biological objects of great significance such as viruses with a tail or fibrils. Compared to top-down beam resonators with electrical read-out and state-of-the-art mass resolution, we show a three-fold improvement in capture area with no resolution degradation, despite the use of a single resonance mode.


Assuntos
Espectrometria de Massas/métodos , Nanotecnologia/métodos , Dispositivos Ópticos , Imagem Individual de Molécula/métodos , Amiloide/química , Desenho de Equipamento , Espectrometria de Massas/instrumentação , Nanopartículas/química , Nanotecnologia/instrumentação , Imagem Individual de Molécula/instrumentação , Vírus/química
6.
Nat Commun ; 11(1): 3607, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32680983

RESUMO

To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of locking on to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART is applied to actively track single Atto 647 N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking is further applied to single proteins and nucleic acids, directly measuring the diffusion of various lengths (99 to 1385 bp) of single DNA molecules at rates up to 10 µm2/s. In addition, 3D-SMART is able to quantify the occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.


Assuntos
DNA/química , Proteínas/química , Imagem Individual de Molécula/métodos , Imagem Individual de Molécula/instrumentação
7.
Mol Cell ; 79(2): 293-303.e4, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32679076

RESUMO

Liquid-liquid phase-separated (LLPS) states are key to compartmentalizing components in the absence of membranes; however, it is unclear whether LLPS condensates are actively and specifically organized in the subcellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB), and a motor (ParA). We show that parS and ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favored by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates, and localizes non-canonical LLPS condensates in the subcellular space.


Assuntos
Trifosfato de Adenosina/fisiologia , Fenômenos Fisiológicos Bacterianos , Proteínas de Escherichia coli/fisiologia , Transição de Fase , DNA Primase/fisiologia , DNA Bacteriano , Microscopia/métodos , Nanopartículas , Imagem Individual de Molécula/métodos
8.
Nat Commun ; 11(1): 3388, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636396

RESUMO

Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.


Assuntos
Corantes Fluorescentes/química , Hidrogéis/química , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Animais , Tampões (Química) , Células COS , Centríolos/metabolismo , Chlamydomonas reinhardtii/metabolismo , Chlorocebus aethiops , Simulação por Computador , Eletrólitos , Epitopos , Imageamento Tridimensional , Microtúbulos/metabolismo , Distribuição Normal , Fotoquímica
9.
Science ; 368(6498): 1449-1454, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32587015

RESUMO

Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/química , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Imagem Individual de Molécula/métodos , Animais , DNA/química , DNA/genética , Drosophila melanogaster , Humanos , Células K562 , Nucleossomos/química , Regiões Promotoras Genéticas , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Fatores de Transcrição/química
10.
Proc Natl Acad Sci U S A ; 117(25): 13937-13944, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513734

RESUMO

Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ∼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.


Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/ultraestrutura , Imagem Individual de Molécula/métodos , Proteínas de Bactérias/ultraestrutura , Caulobacter crescentus/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Transporte Proteico
11.
Nat Commun ; 11(1): 3114, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561744

RESUMO

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3-20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy.


Assuntos
Afinidade de Anticorpos , Epitopos/ultraestrutura , Imunoglobulina G/ultraestrutura , Sondas Moleculares/ultraestrutura , Imagem Individual de Molécula/métodos , Complexo Antígeno-Anticorpo/ultraestrutura , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Epitopos/imunologia , Epitopos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Sondas Moleculares/imunologia , Sondas Moleculares/metabolismo , Nanotecnologia , Relação Estrutura-Atividade
12.
Nat Methods ; 17(7): 734-740, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541853

RESUMO

An outstanding challenge in single-molecule localization microscopy is the accurate and precise localization of individual point emitters in three dimensions in densely labeled samples. One established approach for three-dimensional single-molecule localization is point-spread-function (PSF) engineering, in which the PSF is engineered to vary distinctively with emitter depth using additional optical elements. However, images of dense emitters, which are desirable for improving temporal resolution, pose a challenge for algorithmic localization of engineered PSFs, due to lateral overlap of the emitter PSFs. Here we train a neural network to localize multiple emitters with densely overlapping Tetrapod PSFs over a large axial range. We then use the network to design the optimal PSF for the multi-emitter case. We demonstrate our approach experimentally with super-resolution reconstructions of mitochondria and volumetric imaging of fluorescently labeled telomeres in cells. Our approach, DeepSTORM3D, enables the study of biological processes in whole cells at timescales that are rarely explored in localization microscopy.


Assuntos
Aprendizado Profundo , Imageamento Tridimensional/métodos , Imagem Individual de Molécula/métodos , Fenômenos Biológicos , Redes Neurais de Computação , Telômero/ultraestrutura
13.
Science ; 368(6496): 1253-1257, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32527832

RESUMO

Transition paths of macromolecular conformational changes such as protein folding are predicted to be heterogeneous. However, experimental characterization of the diversity of transition paths is extremely challenging because it requires measuring more than one distance during individual transitions. In this work, we used fast three-color single-molecule Förster resonance energy transfer spectroscopy to obtain the distribution of binding transition paths of a disordered protein. About half of the transitions follow a path involving strong non-native electrostatic interactions, resulting in a transition time of 300 to 800 microseconds. The remaining half follow more diverse paths characterized by weaker electrostatic interactions and more than 10 times shorter transition path times. The chain flexibility and non-native interactions make diverse binding pathways possible, allowing disordered proteins to bind faster than folded proteins.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Intrinsicamente Desordenadas/química , Dobramento de Proteína , Imagem Individual de Molécula/métodos , Ligação Proteica , Conformação Proteica , Eletricidade Estática
14.
Nat Commun ; 11(1): 2828, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504003

RESUMO

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.


Assuntos
DNA de Cadeia Simples/metabolismo , RNA Polimerase III/metabolismo , Imagem Individual de Molécula/métodos , Fator de Transcrição TFIIIB/metabolismo , Transcrição Genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Cinética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Sondas Moleculares/ultraestrutura , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Estabilidade Proteica , RNA Polimerase III/química , Proteínas Recombinantes/metabolismo , Proteína de Ligação a TATA-Box/metabolismo
15.
Mol Cell ; 79(1): 115-126.e6, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32497497

RESUMO

Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combine CRISPR genome editing and MS2 RNA tagging to image single molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, and then base pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life cycle of telomerase RNA and provides a framework to reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis.


Assuntos
Corpos Enovelados/metabolismo , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Imagem Individual de Molécula/métodos , Telomerase/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples/genética , Edição de Genes , Células HeLa , Humanos , Mutação , RNA/genética , Telomerase/genética , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
16.
Mol Genet Genomics ; 295(5): 1227-1237, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32524299

RESUMO

The silk gland of the silkworm Bombyx mori is a specialized organ where silk proteins are efficiently synthesized under precise regulation that largely determines the properties of silk fibers. To understand the genes involved in the regulation of silk protein synthesis, considerable research has focused on the transcripts expressed in silk glands; however, the complete transcriptome profile of this organ has yet to be elucidated. Here, we report a full-length silk gland transcriptome obtained by PacBio single-molecule long-read sequencing technology. In total, 11,697 non-redundant transcripts were identified in mixed samples of silk glands dissected from larvae at five developmental stages. When compared with the published reference, the full-length transcripts optimized the structures of 3002 known genes, and a total of 9061 novel transcripts with an average length of 2171 bp were detected. Among these, 1403 (15.5%) novel transcripts were computationally revealed to be lncRNAs, 8135 (89.8%) novel transcripts were annotated to different protein and nucleotide databases, and 5655 (62.4%) novel transcripts were predicted to have complete ORFs. Furthermore, we found 1867 alternative splicing events, 2529 alternative polyadenylation events, 784 fusion events and 6596 SSRs. This study provides a comprehensive set of reference transcripts and greatly revises and expands the available silkworm transcript data. In addition, these data will be very useful for studying the regulatory mechanisms of silk protein synthesis.


Assuntos
Bombyx/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Seda/genética , Imagem Individual de Molécula/métodos , Processamento Alternativo , Animais , Bombyx/genética , Regulação da Expressão Gênica no Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Insetos/genética , Fases de Leitura Aberta , Poliadenilação , RNA Longo não Codificante/genética , Sequenciamento Completo do Exoma
17.
Nat Methods ; 17(5): 531-540, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371980

RESUMO

Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets, and therefore allows their locations to be pinpointed with precision that achieves the Cramér-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures, from mitochondrial networks and nuclear pores in mammalian cells to amyloid-ß plaques and dendrites in brain tissues and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extracellular targets deep inside tissues.


Assuntos
Encéfalo/metabolismo , Cartilagem/metabolismo , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Placa Amiloide/metabolismo , Imagem Individual de Molécula/métodos , Animais , Encéfalo/patologia , Cartilagem/patologia , Núcleo Celular/metabolismo , Células Cultivadas , Interpretação de Imagem Assistida por Computador/métodos , Masculino , Camundongos , Mitocôndrias/metabolismo , Imagem Molecular/métodos , Poro Nuclear/metabolismo , Placa Amiloide/patologia
18.
Mol Cell ; 79(1): 99-114.e9, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32445620

RESUMO

Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome-bound DNA molecules. These observations shed light on critical processes involved in genome organization in human cells.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula/métodos
19.
Proc Natl Acad Sci U S A ; 117(21): 11257-11264, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404423

RESUMO

Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Saccharomyces cerevisiae Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5'-to-3' polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein-protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Citoesqueleto/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Nucleoproteínas/metabolismo , Rad51 Recombinase/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Imagem Individual de Molécula/métodos
20.
PLoS One ; 15(5): e0232540, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374767

RESUMO

Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®. This is a compact, two-lens illumination system with a constant power objective lens, without any energy filters or aberration correctors, often thought of as a "screening cryo-microscope". Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We used a quasi-crystallographic reciprocal space approach to fit model coordinates to the experimental cryo-EM map. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (X-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving high resolution. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging can be used to determine structures approaching atomic resolution.


Assuntos
Apoferritinas/química , Apoferritinas/ultraestrutura , Microscopia Crioeletrônica/métodos , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica/instrumentação , Cristalografia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Subunidades Proteicas , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Eletricidade Estática
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