RESUMEN
Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1-3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES4-6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55-70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL-GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL-GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.
Asunto(s)
Chaperonina 10 , Chaperonina 60 , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Proteínas de Escherichia coli , Escherichia coli , Sitios de Unión , Chaperonina 10/metabolismo , Chaperonina 10/química , Chaperonina 10/ultraestructura , Chaperonina 60/metabolismo , Chaperonina 60/química , Chaperonina 60/ultraestructura , Citosol/química , Citosol/metabolismo , Citosol/ultraestructura , Escherichia coli/química , Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Modelos Moleculares , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Reproducibilidad de los Resultados , Especificidad por Sustrato , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestructuraRESUMEN
A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic "silver bullet" for improving the quality of every cryoEM sample preparation.
Asunto(s)
Apoferritinas/ultraestructura , Chaperonina 60/ultraestructura , Microscopía por Crioelectrón/métodos , Imagenología Tridimensional/métodos , Proteínas Mitocondriales/ultraestructura , Proteínas Ribosómicas/ultraestructura , Ribosomas/ultraestructura , Aire/análisis , Animales , Biomarcadores/metabolismo , Microscopía por Crioelectrón/instrumentación , Escherichia coli/química , Expresión Génica , Caballos , Humanos , Imagenología Tridimensional/instrumentación , Propiedades de Superficie , Factores de Tiempo , Vitrificación , Agua/químicaRESUMEN
Chaperonins are ubiquitously present protein complexes, which assist the proper folding of newly synthesized proteins and prevent aggregation of denatured proteins in an ATP-dependent manner. They are classified into group I (bacterial, mitochondrial, chloroplast chaperonins) and group II (archaeal and eukaryotic cytosolic variants). However, both of these groups do not include recently discovered viral chaperonins. Here, we solved the symmetry-free cryo-EM structures of a single-ring chaperonin encoded by the gene 246 of bacteriophage OBP Pseudomonas fluorescens, in the nucleotide-free, ATPγS-, and ADP-bound states, with resolutions of 4.3 Å, 5.0 Å, and 6 Å, respectively. The structure of OBP chaperonin reveals a unique subunit arrangement, with three pairs of subunits and one unpaired subunit. Each pair combines subunits in two possible conformations, differing in nucleotide-binding affinity. The binding of nucleotides results in the increase of subunits' conformational variability. Due to its unique structural and functional features, OBP chaperonin can represent a new group.
Asunto(s)
Chaperonina 60/química , Chaperoninas/ultraestructura , Microscopía por Crioelectrón , Chaperonina 60/ultraestructura , Chaperoninas/química , Conformación Proteica , Pliegue de Proteína , Subunidades de Proteína/químicaRESUMEN
BACKGROUND: Cryo-electron tomography (Cryo-ET) is an imaging technique used to generate three-dimensional structures of cellular macromolecule complexes in their native environment. Due to developing cryo-electron microscopy technology, the image quality of three-dimensional reconstruction of cryo-electron tomography has greatly improved. However, cryo-ET images are characterized by low resolution, partial data loss and low signal-to-noise ratio (SNR). In order to tackle these challenges and improve resolution, a large number of subtomograms containing the same structure needs to be aligned and averaged. Existing methods for refining and aligning subtomograms are still highly time-consuming, requiring many computationally intensive processing steps (i.e. the rotations and translations of subtomograms in three-dimensional space). RESULTS: In this article, we propose a Stochastic Average Gradient (SAG) fine-grained alignment method for optimizing the sum of dissimilarity measure in real space. We introduce a Message Passing Interface (MPI) parallel programming model in order to explore further speedup. CONCLUSIONS: We compare our stochastic average gradient fine-grained alignment algorithm with two baseline methods, high-precision alignment and fast alignment. Our SAG fine-grained alignment algorithm is much faster than the two baseline methods. Results on simulated data of GroEL from the Protein Data Bank (PDB ID:1KP8) showed that our parallel SAG-based fine-grained alignment method could achieve close-to-optimal rigid transformations with higher precision than both high-precision alignment and fast alignment at a low SNR (SNR=0.003) with tilt angle range ±60∘ or ±40∘. For the experimental subtomograms data structures of GroEL and GroEL/GroES complexes, our parallel SAG-based fine-grained alignment can achieve higher precision and fewer iterations to converge than the two baseline methods.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Chaperonina 10/ultraestructura , Chaperonina 60/ultraestructura , Bases de Datos de Proteínas , Procesamiento de Imagen Asistido por Computador/métodos , Relación Señal-Ruido , Factores de TiempoRESUMEN
Electron cryotomography enables 3D visualization of cells in a near-native state at molecular resolution. The produced cellular tomograms contain detailed information about a plethora of macromolecular complexes, their structures, abundances, and specific spatial locations in the cell. However, extracting this information in a systematic way is very challenging, and current methods usually rely on individual templates of known structures. Here, we propose a framework called "Multi-Pattern Pursuit" for de novo discovery of different complexes from highly heterogeneous sets of particles extracted from entire cellular tomograms without using information of known structures. These initially detected structures can then serve as input for more targeted refinement efforts. Our tests on simulated and experimental tomograms show that our automated method is a promising tool for supporting large-scale template-free visual proteomics analysis.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Chaperonina 60/ultraestructura , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Proteínas Bacterianas/metabolismo , Bdellovibrio bacteriovorus/metabolismo , Bdellovibrio bacteriovorus/ultraestructura , Chaperonina 60/metabolismo , Comamonadaceae/metabolismo , Comamonadaceae/ultraestructura , Microscopía por Crioelectrón/instrumentación , Minería de Datos , Tomografía con Microscopio Electrónico/instrumentación , Firmicutes/metabolismo , Firmicutes/ultraestructura , Imagenología Tridimensional , ProteómicaRESUMEN
The chloroplast chaperonin system is indispensable for the biogenesis of Rubisco, the key enzyme in photosynthesis. Using Chlamydomonas reinhardtii as a model system, we found that in vivo the chloroplast chaperonin consists of CPN60α, CPN60ß1 and CPN60ß2 and the co-chaperonin of the three subunits CPN20, CPN11 and CPN23. In Escherichia coli, CPN20 homo-oligomers and all possible other chloroplast co-chaperonin hetero-oligomers are functional, but only that consisting of CPN11/20/23-CPN60αß1ß2 can fully replace GroES/GroEL under stringent stress conditions. Endogenous CPN60 was purified and its stoichiometry was determined to be 6:2:6 for CPN60α:CPN60ß1:CPN60ß2. The cryo-EM structures of endogenous CPN60αß1ß2/ADP and CPN60αß1ß2/co-chaperonin/ADP were solved at resolutions of 4.06 and 3.82 Å, respectively. In both hetero-oligomeric complexes the chaperonin subunits within each ring are highly symmetric. Through hetero-oligomerization, the chloroplast co-chaperonin CPN11/20/23 forms seven GroES-like domains, which symmetrically interact with CPN60αß1ß2. Our structure also reveals an uneven distribution of roof-forming domains in the dome-shaped CPN11/20/23 co-chaperonin and potentially diversified surface properties in the folding cavity of the CPN60αß1ß2 chaperonin that might enable the chloroplast chaperonin system to assist in the folding of specific substrates.
Asunto(s)
Chaperonina 60/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Chaperoninas del Grupo I/metabolismo , Chaperonina 60/química , Chaperonina 60/ultraestructura , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/ultraestructura , Cloroplastos/ultraestructura , Microscopía por Crioelectrón/métodos , Chaperoninas del Grupo I/química , Chaperoninas del Grupo I/ultraestructura , Fotosíntesis , Pliegue de Proteína , Multimerización de Proteína , Subunidades de Proteína/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismoRESUMEN
The products of the reassembly reaction of tetradecameric two-ring quaternary structure of GroEL chaperonin under the pressure of its heptameric co-chaperonin GroES have been visualized by electron microscopy. It has been shown that one-ring heptameric oligomers of GroEL have been formed at the beginning (after ~5 min) of the reaction, while at the final stage of the reaction (after ~70 min), both one-ring heptamers in complex with one GroES and two-rings tetradecamers in complexes with one (asymmetrical complex) or two (symmetrical complex) GroES heptamers are present. The relationship between the data of light scattering, native electrophoresis, and electron microscopy obtained earlier has been discussed.
Asunto(s)
Chaperonina 10/química , Chaperonina 60/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Chaperonina 10/ultraestructura , Chaperonina 60/ultraestructura , Proteínas de Escherichia coli/ultraestructura , Microscopía Electrónica , Unión Proteica , Pliegue de ProteínaRESUMEN
We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with Nα, Nα-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 Å. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM.
Asunto(s)
Chaperonina 60/ultraestructura , Proteínas de Escherichia coli/ultraestructura , Histidina/química , Lisina/análogos & derivados , Microscopía Electrónica de Transmisión/instrumentación , Oligopéptidos/química , Proteínas Recombinantes de Fusión/química , Ácido 4-Aminobenzoico/química , Bacteriófago T7/química , Bacteriófago T7/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Grafito/química , Histidina/metabolismo , Lisina/química , Membranas Artificiales , Oligopéptidos/metabolismo , Óxidos/química , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
CryoEM single-particle reconstruction has been growing rapidly over the last 3 years largely due to the development of direct electron detectors, which have provided data with dramatic improvements in image quality. It is now possible in many cases to produce near-atomic resolution structures, and yet 2/3 of published structures remain at substantially lower resolutions. One important cause for this is compositional and conformational heterogeneity, which is both a resolution-limiting factor and presenting a unique opportunity to better relate structure to function. This manuscript discusses the canonical methods for high-resolution refinement in EMAN2.12, and then considers the wide range of available methods within this package for resolving structural variability, targeting both improved resolution and additional knowledge about particle dynamics.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Análisis de Varianza , Artefactos , Proteínas Bacterianas/ultraestructura , Chaperonina 60/ultraestructura , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/métodos , Movimiento (Física) , Ribosomas/ultraestructuraRESUMEN
As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5-4.5Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders' overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite.
Asunto(s)
Imagenología Tridimensional , Programas Informáticos , Adenilato Quinasa/química , Adenilato Quinasa/ultraestructura , Chaperonina 60/química , Chaperonina 60/ultraestructura , Microscopía por Crioelectrón , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Factores Eucarióticos de Iniciación/química , Factores Eucarióticos de Iniciación/ultraestructura , Modelos Moleculares , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructuraRESUMEN
Dynamics can provide deep insights into the functional mechanisms of proteins and protein complexes. For large protein complexes such as GroEL/GroES with more than 8,000 residues, obtaining a fine-grained all-atom description of its normal mode motions can be computationally prohibitive and is often unnecessary. For this reason, coarse-grained models have been used successfully. However, most existing coarse-grained models use extremely simple potentials to represent the interactions within the coarse-grained structures and as a result, the dynamics obtained for the coarse-grained structures may not always be fully realistic. There is a gap between the quality of the dynamics of the coarse-grained structures given by all-atom models and that by coarse-grained models. In this work, we resolve an important question in protein dynamics computations--how can we efficiently construct coarse-grained models whose description of the dynamics of the coarse-grained structures remains as accurate as that given by all-atom models? Our method takes advantage of the sparseness of the Hessian matrix and achieves a high efficiency with a novel iterative matrix projection approach. The result is highly significant since it can provide descriptions of normal mode motions at an all-atom level of accuracy even for the largest biomolecular complexes. The application of our method to GroEL/GroES offers new insights into the mechanism of this biologically important chaperonin, such as that the conformational transitions of this protein complex in its functional cycle are even more strongly connected to the first few lowest frequency modes than with other coarse-grained models.
Asunto(s)
Chaperonina 10/química , Chaperonina 10/ultraestructura , Chaperonina 60/química , Chaperonina 60/ultraestructura , Modelos Químicos , Simulación del Acoplamiento Molecular/métodos , Sitios de Unión , Módulo de Elasticidad , Movimiento (Física) , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas/métodosRESUMEN
The Chaperonins comprise a family of molecular chaperones having a double-ring structure and similar sequence homology. These proteins play an essential role in biological reactions that mediate the folding of newly synthesized polypeptides and partially denatured proteins. In the prokaryotic group I chaperonins, structural and reaction cycle analyses of GroEL and its co-chaperone GroES have been performed in detail. While in eukaryotes, there have been limited reports analyzing the group I chaperonin HSP60 and its co-chaperone HSP10. In the present study, we purified the wild type HSP60 from porcine liver and investigated the interaction between HSP60 and HSP10, including conformation and physiological relationships. Based on the results of transmission electron microscopy, native PAGE, and gel filtration column chromatography, the wild type HSP60 displayed a heptameric single-ring structure in the absence of ATP. In contrast, HSP60 formed mainly a "football-type" complex with HSP10 in the presence of ATP and mediated the refolding of denatured substrate protein. The functional conformation cycle of the purified mammalian HSP60 is distinct from the cycle of the prokaryotic GroEL/GroES chaperonin.
Asunto(s)
Chaperonina 60/química , Chaperonina 60/fisiología , Adenosina Trifosfato/metabolismo , Animales , Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/ultraestructura , Técnicas In Vitro , Cinética , Microscopía Electrónica de Transmisión , Conformación Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Sus scrofaRESUMEN
Bacterial toxin or viral entry into the cell often requires cell surface binding and endocytosis. The endosomal acidification induces a limited unfolding/refolding and membrane insertion reaction of the soluble toxins or viral proteins into their translocation competent or membrane inserted states. At the molecular level, the specific orientation and immobilization of the pre-transitioned toxin on the cell surface is often an important prerequisite prior to cell entry. We propose that structures of some toxin membrane insertion complexes may be observed through procedures where one rationally immobilizes the soluble toxin so that potential unfolding â refolding transitions that occur prior to membrane insertion orientate away from the immobilization surface in the presence of lipid micelle pre-nanodisc structures. As a specific example, the immobilized prepore form of the anthrax toxin pore translocon or protective antigen can be transitioned, inserted into a model lipid membrane (nanodiscs), and released from the immobilized support in its membrane solubilized form. This particular strategy, although unconventional, is a useful procedure for generating pure membrane-inserted toxins in nanodiscs for electron microscopy structural analysis. In addition, generating a similar immobilized platform on label-free biosensor surfaces allows one to observe the kinetics of these acid-induced membrane insertion transitions. These platforms can facilitate the rational design of inhibitors that specifically target the toxin membrane insertion transitions that occur during endosomal acidification. This approach may lead to a new class of direct anti-toxin inhibitors.
Asunto(s)
Membrana Dobles de Lípidos/química , Antígenos Bacterianos/ultraestructura , Toxinas Bacterianas , Técnicas Biosensibles , Membrana Celular/química , Membrana Celular/ultraestructura , Chaperonina 60/ultraestructura , Microscopía por Crioelectrón , Endosomas/química , Modelos Moleculares , Nanoestructuras/química , Nanoestructuras/ultraestructura , Estructura Terciaria de ProteínaRESUMEN
The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was â¼ 20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of â¼ 150 h (â¼ 6 days), providing a good model to characterize the football-shaped complex.
Asunto(s)
Adenosina Trifosfato/metabolismo , Ácido Aspártico/metabolismo , Chaperonina 60/metabolismo , Escherichia coli/metabolismo , Sitios de Unión , Chaperonina 10/metabolismo , Chaperonina 10/ultraestructura , Chaperonina 60/química , Chaperonina 60/ultraestructura , Hidrólisis , Malato Deshidrogenasa/metabolismo , Proteínas Mutantes/metabolismo , Coloración Negativa , Pliegue de Proteína , Estabilidad Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Tiosulfato Azufretransferasa/metabolismoRESUMEN
Protein nanoassemblies possess unique advantage in biomedical applications such as drug delivery, biocatalysis and vaccine development. Despite recent accomplishment in atomic structure data, the underlying molecular mechanism of protein self-assembly remains elusive, where considerable heterogeneity is often involved. Here we use E. coli chaperonin GroEL, a tetradecameric protein with a molecular weight of 805 kDa, to probe its transformation from cage-like oligomers to protein nanofibers. We show that sodium dodecyl sulfate (SDS), a widely-used protein denaturant, at submicellar concentration binds to and causes partial distortion of GroEL apical domain. Subsequently, the GroEL apical domain with altered secondary structural content converts the GroEL oligomers into modular structural units which are observed to self-assemble into cylindrical nanofibers under an agitated incubation in a physiological buffer. Interestingly, through targeted mutagenesis where two cysteine residues are introduced at the entry site of GroEL cage, we found that the formation of GroEL nanoassembly could be modulated depending on the redox condition of incubation. Without the need of chemical engineering, tunable GroEL nanofibers built by controlled-assembly are among the largest nanoscale bioassembly with broad applications.
Asunto(s)
Chaperonina 60/química , Chaperonina 60/ultraestructura , Nanofibras/química , Nanofibras/ultraestructura , Dodecil Sulfato de Sodio/química , Sitios de Unión , Cristalización/métodos , Micelas , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Unión ProteicaRESUMEN
One single-particle reconstruction technique is the reconstruction of macromolecules from projection images of randomly oriented particles (SPRR). In SPRR the reliability and consequent interpretation of the final reconstruction is affected by errors arising from incorrect assignment of projection angles to individual particles. In order to improve the resolution of SPRR we studied the influence of imperfect assignment on 3D blurring. We find that this blurring can be described as a Point Spread Function (PSF) that depends on the distance from geometrical center of the reconstructed volume and that blurring is higher at the periphery. This particular PSF can be described by an almost pure tangential angular function with a negligible radial component. We have developed a reliable algorithm for spherical deconvolution of the 3D reconstruction. This spherical deconvolution operation was tested on reconstructions of GroEL and mitochondrial ribosomes. We show that spherical deconvolution improves the quality of SPRR by reducing blurring and enhancing high frequency components, particularly near the periphery of the reconstruction.
Asunto(s)
Algoritmos , Proteínas Bacterianas/ultraestructura , Chaperonina 60/ultraestructura , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Ribosomas/ultraestructura , Microscopía por Crioelectrón , Imagenología Tridimensional/estadística & datos numéricos , Mitocondrias/química , Modelos MolecularesRESUMEN
Cryo-electron tomography provides a snapshot of the cellular proteome. With template matching, the spatial positions of various macromolecular complexes within their native cellular context can be detected. However, the growing awareness of the reference bias introduced by the cross-correlation based approaches, and more importantly the lack of a reliable confidence measurement in the selection of these macromolecular complexes, has restricted the use of these applications. Here we propose a heuristic, in which the reference bias is measured in real space in an analogous way to the R-free value in X-ray crystallography. We measure the reference bias within the mask used to outline the area of the template, and do not modify the template itself. The heuristic works by splitting the mask into a working and a testing area in a volume ratio of 9:1. While the working area is used during the calculation of the cross-correlation function, the information from both areas is explored to calculate the M-free score. We show using artificial data, that the M-free score gives a reliable measure for the reference bias. The heuristic can be applied in template matching and in sub-tomogram averaging. We further test the applicability of the heuristic in tomograms of purified macromolecules, and tomograms of whole Mycoplasma cells.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Chaperonina 60/ultraestructura , Microscopía por Crioelectrón/estadística & datos numéricos , Tomografía con Microscopio Electrónico/estadística & datos numéricos , Mycoplasma/ultraestructura , Algoritmos , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Teoría Cuántica , Proyectos de Investigación , TermodinámicaRESUMEN
Here, we employed the collective motions extracted from Normal Mode Analysis (NMA) in internal coordinates (torsional space) for the flexible fitting of atomic-resolution structures into electron microscopy (EM) density maps. The proposed methodology was validated using a benchmark of simulated cases, highlighting its robustness over the full range of EM resolutions and even over coarse-grained representations. A systematic comparison with other methods further showcased the advantages of this proposed methodology, especially at medium to lower resolutions. Using this method, computational costs and potential overfitting problems are naturally reduced by constraining the search in low-frequency NMA space, where covalent geometry is implicitly maintained. This method also effectively captures the macromolecular changes of a representative set of experimental test cases. We believe that this novel approach will extend the currently available EM hybrid methods to the atomic-level interpretation of large conformational changes and their functional implications.
Asunto(s)
Microscopía por Crioelectrón , Programas Informáticos , Algoritmos , Chaperonina 60/química , Chaperonina 60/ultraestructura , Simulación por Computador , Interpretación Estadística de Datos , Microtúbulos/química , Microtúbulos/ultraestructura , Modelos Moleculares , Estructura Cuaternaria de Proteína , Ribosomas/química , Ribosomas/ultraestructuraRESUMEN
Fitting high resolution protein structures into low resolution cryo-electron microscopy (cryo-EM) density maps is an important technique for modeling the atomic structures of very large macromolecular assemblies. This article presents "gEMfitter", a highly parallel fast Fourier transform (FFT) EM density fitting program which can exploit the special hardware properties of modern graphics processor units (GPUs) to accelerate both the translational and rotational parts of the correlation search. In particular, by using the GPU's special texture memory hardware to rotate 3D voxel grids, the cost of rotating large 3D density maps is almost completely eliminated. Compared to performing 3D correlations on one core of a contemporary central processor unit (CPU), running gEMfitter on a modern GPU gives up to 26-fold speed-up. Furthermore, using our parallel processing framework, this speed-up increases linearly with the number of CPUs or GPUs used. Thus, it is now possible to use routinely more robust but more expensive 3D correlation techniques. When tested on low resolution experimental cryo-EM data for the GroEL-GroES complex, we demonstrate the satisfactory fitting results that may be achieved by using a locally normalised cross-correlation with a Laplacian pre-filter, while still being up to three orders of magnitude faster than the well-known COLORES program.
Asunto(s)
Imagenología Tridimensional , Programas Informáticos , Algoritmos , Chaperonina 60/química , Chaperonina 60/ultraestructura , Microscopía por Crioelectrón , Análisis de Fourier , Modelos Moleculares , Estructura Cuaternaria de Proteína , Rec A Recombinasas/química , Rec A Recombinasas/ultraestructuraRESUMEN
We present a map-restrained self-guided Langevin dynamics (MapSGLD) simulation method for efficient targeted conformational search. The targeted conformational search represents simulations under restraints defined by experimental observations and/or by user specified structural requirements. Through map-restraints, this method provides an efficient way to maintain substructures and to set structure targets during conformational searching. With an enhanced conformational searching ability of self-guided Langevin dynamics, this approach is suitable for simulating large-scale conformational changes, such as the formation of macromolecular assemblies and transitions between different conformational states. Using several examples, we illustrate the application of this method in flexible fitting of atomic structures into density maps derived from cryo-electron microscopy.