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1.
The extracellular giant hemoglobin from the earthworm Lumbricus terrestris was reconstructed at 14.9-Å resolution from cryo-electron microscope images, using a new procedure for estimating parameters of the contrast transfer (CTF) function. In this approach, two important CTF parameters, defocus and amplitude contrast ratio, can be refined iteratively within the framework of 3D projection alignment procedure, using minimization of sign disagreement between theoretical CTF and cross-resolution curves. The 3D cryo-EM map is in overall good agreement with the recent X-ray crystallography map of Royer et al. (2000, Proc. Natl. Acad. Sci. USA 97, 7107–7111), and it reveals the local threefold arrangement of the three linker chains present within each 1/12 of the complex. The 144 globin chains and 36 linker chains within the complex are clearly visible, and the interdigitation of the 12 coiled-coil helical spokes forming the central toroidal piece is confirmed. Based on these findings, two mechanisms of the dodecameric unit assembly are proposed and termed “zigzag” and “pairwise” polymerizations. However, the detection by cryo-EM of 12 additional rod-like bodies within the toroid raises the possibility that the architecture of the toroid is more complex than previously thought or that yet unknown ligands or allosteric effectors for this oxygen carrier are present. 相似文献
2.
Min Su 《Journal of structural biology》2019,205(1):22-29
Preferred particle orientation represents a recurring problem in single-particle cryogenic electron microcopy (cryo-EM). A specimen-independent approach through tilting has been attempted to increase particle orientation coverage, thus minimizing anisotropic three-dimensional (3D) reconstruction. However, focus gradient is a critical issue hindering tilt applications from being a general practice in single-particle cryo-EM. The present study describes a newly developed geometrically optimized approach, goCTF, to reliably determine the global focus gradient. A novel strategy of determining contrast transfer function (CTF) parameters from a sector of the signal preserved power spectrum is applied to increase reliability. Subsequently, per-particle based local focus refinement is conducted in an iterative manner to further improve the defocus accuracy. Novel diagnosis methods using a standard deviation defocus plot and goodness of fit heatmap have also been proposed to evaluate CTF fitting quality prior to 3D refinement. In a benchmark study, goCTF processed a published single-particle cryo-EM dataset for influenza hemagglutinin trimer collected at a 40-degree specimen tilt. The resulting 3D reconstruction map was improved from 4.1?Å to 3.7?Å resolution. The goCTF program is built on the open-source code of CTFFIND4, which adopts a consistent user interface for ease of use. 相似文献
3.
Evidence that aspartate-85 has a higher pK(a) in all-trans than in 13-cisbacteriorhodopsin.
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Macrobdella decora hemoglobin was observed in vitreous ice by cryoelectron microscopy and subjected to three-dimensional reconstruction by the method of random conical tilt series. The refined volume has a resolution of 40 A and a D6 point-group symmetry. Its architecture, with its hexagonal bilayer appearance, resembles those of Lumbricus terrestris (oligochaete) and Eudistylia vancouverii (polychaete). When the reconstruction volume is viewed along its sixfold axis, the vertices of the upper hexagonal layer are rotated 16 degrees clockwise compared to those of the lower layer. In agreement with the "bracelet" model of Vinogradov et al., a central linker complex is decorated by 12 hollow globular substructures. The linker complex is made up of a central hexagonal toroid linked by 12 c5 connections to two bracelets of c3 connections, which are themselves linked via six c4 connections. The portion of the hollow globular substructure corresponding to the dodecamer of globin chains has a local pseudo threefold symmetry and is composed of three elongated structures visible when the volume is displayed at high threshold. The main difference between Macrobdella, Lumbricus, and Eudistylia hemoglobins is the presence in Macrobdella of a central hexagonal toroid instead of a compact flat hexagonal structure. 相似文献
4.
A 3D reconstruction of the hemoglobin (Hb) of the earthworm Lumbricus terrestris was carried out by the 3D projection alignment method from electron microscopy images of a frozen-hydrated specimen at 22 A resolution. The results were analyzed by a new approach taking into account the evolution of the 210 densities forming the 3D volume as a function of the threshold of surface representation. The whole oligomer with D6point-group symmetry is comprised of 12 hollow globular substructures (HGS) with local 3-fold symmetry tethered to a complex network of linking subunits (linker complex). The 12 globin subunits of each HGS are distributed around local 3-fold axis in four layers of three subunits. The first layer, the most external, contains monomeric globin chains 2A, 3A, and 5A. The three trimers corresponding to the nine remaining subunits have one subunit in each of the second (2B, 3B, 5B), third (1A, 4A, 6A), and fourth (1B, 4B, 6B) layer. The distances between the centers of the globin chains forming the trimers are in the ranges 20-32 A and 45-52 A. The linker complex is made up of two types of linking units. The first type forms three loops connecting globin chains of the second, third and fourth layers. The average molecular mass (Mm) of these subunits was 25 kDa. The second type forms the central structure, termed hexagonal toroid, and its 12 connections to the HGS. This structure corresponds to a hexamer of a single linking unit with a Mm (31.2 kDa), size and a shape different from those of the HGS loops. A careful study of 3D volume architecture shows that each toroid linking unit is bound to the three loops of a HGS pair located in the upper and lower hexagonal layers, respectively. As shown in a model of architecture, hexagonal bilayered (HBL) Hbs can be built very simply from 144 globin chains and 42 linker chains belonging to two different types. We also propose a simple assembly sequence for the construction of HBL Hbs based on the architecture model. 相似文献
5.
Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly. 相似文献
6.
7.
A frozen-hydrated specimen of the V1 hemoglobin of the hydrothermal vent tube worm Riftia pachyptila was observed in the electron microscope and subjected to three-dimensional reconstruction by the method of random conical tilt series. The 3D volume possesses a D6 point-group symmetry. When viewed along its 6-fold axis the vertices of its upper hexagonal layer are 16° clockwise rotated compared to those of the lower layer. A central linker complex is decorated by 12 hollow globular substructures. The linker complex comprises (i) a central hexagonal toroid, (ii) two internal bracelets onto which the hollow globular substructures are built, and (iii) six structures connecting the two hexagonal layers. The hollow globular substructures, related to the dodecamers of globin chains resulting from the dissociation of the hexagonal bilayer hemoglobin, have a local pseudo 3-fold symmetry and are composed each of three elongated structures visible when the volume is displayed at high threshold. At a resolution of 36 Å, the 3D volumes of the hexagonal bilayer hemoglobins of Riftia pachyptyla and of the leech Macrobdella decora look almost perfectly identical. © 1996 Wiley-Liss, Inc. 相似文献
8.
Dodecamers and four types of linker chains (L1-L4) were purified from dissociated hemoglobin of the earthworm Lumbricus terrestris. Various preparations comprising dodecamer of globin chains and linker chains were allowed to reassemble at neutral pH. They produced various oligomers that were purified by gel filtration, analyzed in matrix-assisted laser desorption/ionization mass spectrometry and submitted to 3D reconstruction from isolated particles observed in cryoelectron microscopy. Despite the impossibility to completely free the L2, L3, and L4 preparations from L1, the following conclusions were obtained. First, hemoglobin molecules indistinguishable from native hemoglobin at 25 A resolution were obtained in the absence of linker chains L2, L3, or L4. Second, the 3D reconstruction volumes of reassembled hemoglobins containing dodecamers and L1+L3 or dodecamers and L1+L4 demonstrate that reassembly of native-like structures can be obtained from at most two linker chains and dodecamers. Third, the 3D reconstruction volumes of native and reassembled hemoglobins containing dodecamers and (1) L1, L2, and L4, (2) L1, L3, and L4, (3) L1 and L4, and (4) L1 and L3 were highly similar. Since these structures comprise two types of substructures (one involved in the c3a, c3b, and c4 linking units of the hollow globular substructure and the other in the c5 connection and the toroid), it seems highly probable that the minimal number of linker chains required to reassemble native-like hemoglobin is at most two. 相似文献
9.
Toshiyuki Oda Tatsuki Abe Haruaki Yanagisawa Masahide Kikkawa 《Molecular biology of the cell》2016,27(7):1051-1059
The outer dynein arm (ODA) is a molecular complex that drives the beating motion of cilia/flagella. Chlamydomonas ODA is composed of three heavy chains (HCs), two ICs, and 11 light chains (LCs). Although the three-dimensional (3D) structure of the whole ODA complex has been investigated, the 3D configurations of the ICs and LCs are largely unknown. Here we identified the 3D positions of the two ICs and three LCs using cryo–electron tomography and structural labeling. We found that these ICs and LCs were all localized at the root of the outer-inner dynein (OID) linker, designated the ODA-Beak complex. Of interest, the coiled-coil domain of IC2 extended from the ODA-Beak to the outer surface of ODA. Furthermore, we investigated the molecular mechanisms of how the OID linker transmits signals to the ODA-Beak, by manipulating the interaction within the OID linker using a chemically induced dimerization system. We showed that the cross-linking of the OID linker strongly suppresses flagellar motility in vivo. These results suggest that the ICs and LCs of the ODA form the ODA-Beak, which may be involved in mechanosignaling from the OID linker to the HCs. 相似文献
10.
The bootstrap-based method for calculation of the 3D variance in cryo-EM maps reconstructed from sets of their projections was applied to a dataset of functional ribosomal complexes containing the Escherichia coli 70S ribosome, tRNAs, and elongation factor G (EF-G). The variance map revealed regions of high variability in the intersubunit space of the ribosome: in the locations of tRNAs, in the putative location of EF-G, and in the vicinity of the L1 protein. This result indicated heterogeneity of the dataset. A method of focused classification was put forward in order to sort out the projection data into approximately homogenous subsets. The method is based on the identification and localization of a region of high variance that a subsequent classification step can be focused on by the use of a 3D spherical mask. After initial classification, template volumes are created and are subsequently refined using a multireference 3D projection alignment procedure. In the application to the ribosome dataset, the two resulting structures were interpreted as resulting from ribosomal complexes with bound EF-G and an empty A site, or, alternatively, from complexes that had no EF-G bound but had both A and P sites occupied by tRNA. The proposed method of focused classification proved to be a successful tool in the analysis of the heterogeneous cryo-EM dataset. The associated calculation of the correlations within the density map confirmed the conformational variability of the complex, which could be interpreted in terms of the ribosomal elongation cycle. 相似文献
11.
Accurate knowledge of defocus and tilt parameters is essential for the determination of three-dimensional protein structures at high resolution using electron microscopy. We present two computer programs, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from images of tilted specimens, respectively. Both programs use a simple algorithm that fits the amplitude modulations visible in a power spectrum with a calculated contrast transfer function (CTF). The background present in the power spectrum is calculated using a low-pass filter. The background is then subtracted from the original power spectrum, allowing the fitting of only the oscillatory component of the CTF. CTFTILT determines specimen tilt parameters by measuring the defocus at a series of locations on the image while constraining them to a single plane. We tested the algorithm on images of two-dimensional crystals by comparing the results with those obtained using crystallographic methods. The images also contained contrast from carbon support film that added to the visibility of the CTF oscillations. The tests suggest that the fitting procedure is able to determine the image defocus with an error of about 10nm, whereas tilt axis and tilt angle are determined with an error of about 2 degrees and 1 degrees, respectively. Further tests were performed on images of single protein particles embedded in ice that were recorded from untilted or slightly tilted specimens. The visibility of the CTF oscillations from these images was reduced due to the lack of a carbon support film. Nevertheless, the test results suggest that the fitting procedure is able to determine image defocus and tilt angle with errors of about 100 nm and 6 degrees, respectively. 相似文献
12.
The hemoglobin of the polychaete worm Alvinella pompejana was reconstructed at 20A resolution from frozen-hydrated samples observed by electron microscopy according to the random conical tilt series method. This three-dimensional reconstruction was mirror-inverted with respect to a previous volume published by de Haas et al. in 1996. In order to explain this handedness discrepancy, various 3D reconstructions using different reference volumes were carried out showing that the choice of the first volume was the keystone during the refinement process. The 3D reconstruction volume of A. pompejana Hb presented structural features characteristic of annelid Hbs with two hexagonal layers each comprising six hollow globular subassemblies and a complex of non-heme linker chains. Moreover, the eclipsed conformation of the two hexagonal layers and a HGS architecture similar to that described for Arenicola marina Hb led to the conclusion that A. pompejana Hb belonged to the architectural type II according to the definition of Jouan et al. (2001). A comparison between this cryo-EM volume and X-ray crystallography density maps of Lumbricus terrestris type-I Hb (Royer et al., 2000) showed that the triple stranded coiled coil structures of linker chains were different. Based on this observation, a model was proposed to explain the eclipsed conformation of the two hexagonal layers of type-II Hbs. 相似文献
13.
《Journal of structural biology》2014,185(1):42-47
Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500 nm holes spaced 4 μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently. 相似文献
14.
Three-dimensional (3D) reconstruction in single-particle cryo-electron microscopy (cryo-EM) is a significant technique for recovering the 3D structure of proteins or other biological macromolecules from their two-dimensional (2D) noisy projection images taken from unknown random directions. Class averaging in single-particle cryo-EM is an important procedure for producing high-quality initial 3D structures, where image alignment is a fundamental step. In this paper, an efficient image alignment algorithm using 2D interpolation in the frequency domain of images is proposed to improve the estimation accuracy of alignment parameters of rotation angles and translational shifts between the two projection images, which can obtain subpixel and subangle accuracy. The proposed algorithm firstly uses the Fourier transform of two projection images to calculate a discrete cross-correlation matrix and then performs the 2D interpolation around the maximum value in the cross-correlation matrix. The alignment parameters are directly determined according to the position of the maximum value in the cross-correlation matrix after interpolation. Furthermore, the proposed image alignment algorithm and a spectral clustering algorithm are used to compute class averages for single-particle 3D reconstruction. The proposed image alignment algorithm is firstly tested on a Lena image and two cryo-EM datasets. Results show that the proposed image alignment algorithm can estimate the alignment parameters accurately and efficiently. The proposed method is also used to reconstruct preliminary 3D structures from a simulated cryo-EM dataset and a real cryo-EM dataset and to compare them with RELION. Experimental results show that the proposed method can obtain more high-quality class averages than RELION and can obtain higher reconstruction resolution than RELION even without iteration. 相似文献
15.
16.
We present the codimensional principal component analysis (PCA), a novel and straightforward method for resolving sample heterogeneity within a set of cryo-EM 2D projection images of macromolecular assemblies. The method employs PCA of resampled 3D structures computed using subsets of 2D data obtained with a novel hypergeometric sampling scheme. PCA provides us with a small subset of dominating "eigenvolumes" of the system, whose reprojections are compared with experimental projection data to yield their factorial coordinates constructed in a common framework of the 3D space of the macromolecule. Codimensional PCA is unique in the dramatic reduction of dimensionality of the problem, which facilitates rapid determination of both the plausible number of conformers in the sample and their 3D structures. We applied the codimensional PCA to a complex data set of Thermus thermophilus 70S ribosome, and we identified four major conformational states and visualized high mobility of the stalk base region. 相似文献
17.
Background
Proteins involved in mammalian mitochondrial translation, when compared to analogous bacterial proteins, frequently have additional sequence regions whose structural or functional roles are not always clear. For example, an additional short insert sequence in the bovine mitochondrial initiation factor 2 (IF2mt) seems sufficient to fulfill the added role of eubacterial initiation factor IF1. Prior to our recent cryo-EM study that showed IF2mt to structurally occupy both the IF1 and IF2 binding sites, the spatial separation of these sites, and the short length of the insert sequence, posed ambiguity in whether it could perform the role of IF1 through occupation of the IF1 binding site on the ribosome.Results
The present study probes how well computational structure prediction methods can a priori address hypothesized roles of such additional sequences by creating quasi-atomic models of IF2mt using bacterial IF2 cryo-EM densities (that lack the insert sequences). How such initial IF2mt predictions differ from the observed IF2mt cryo-EM map and how they can be suitably improved using further sequence analysis and flexible fitting are analyzed.Conclusions
By hypothesizing that the insert sequence occupies the IF1 binding site, continuous IF2mt models that occupy both the IF2 and IF1 binding sites can be predicted computationally. These models can be improved by flexible fitting into the IF2mt cryo-EM map to get reasonable quasi-atomic IF2mt models, but the exact orientation of the insert structure may not be reproduced. Specific eukaryotic insert sequence conservation characteristics can be used to predict alternate IF2mt models that have minor secondary structure rearrangements but fewer unusually extended linker regions. Computational structure prediction methods can thus be combined with medium-resolution cryo-EM maps to explore structure-function hypotheses for additional sequence regions and to guide further biochemical experiments, especially in mammalian systems where high-resolution structures are difficult to determine. 相似文献18.
LeBarron J Grassucci RA Shaikh TR Baxter WT Sengupta J Frank J 《Journal of structural biology》2008,164(1):24-32
A number of image processing parameters in the 3D reconstruction of a ribosome complex from a cryo-EM data set were varied to test their effects on the final resolution. The parameters examined were pixel size, window size, and mode of Fourier amplitude enhancement at high spatial frequencies. In addition, the strategy of switching from large to small pixel size during angular refinement was explored. The relationship between resolution (in Fourier space) and the number of particles was observed to follow a lin-log dependence, a relationship that appears to hold for other data, as well. By optimizing the above parameters, and using a lin-log extrapolation to the full data set in the estimation of resolution from half-sets, we obtained a 3D map from 131,599 ribosome particles at 6.7A resolution (FSC=0.5). 相似文献
19.
Cryo-elecron microscopy (cryo-EM) can provide important structural information of large macromolecular assemblies in different conformational states. Recent years have seen an increase in structures deposited in the Protein Data Bank (PDB) by fitting a high-resolution structure into its low-resolution cryo-EM map. A commonly used protocol for accommodating the conformational changes between the X-ray structure and the cryo-EM map is rigid body fitting of individual domains. With the emergence of different flexible fitting approaches, there is a need to compare and revise these different protocols for the fitting. We have applied three diverse automated flexible fitting approaches on a protein dataset for which rigid domain fitting (RDF) models have been deposited in the PDB. In general, a consensus is observed in the conformations, which indicates a convergence from these theoretically different approaches to the most probable solution corresponding to the cryo-EM map. However, the result shows that the convergence might not be observed for proteins with complex conformational changes or with missing densities in cryo-EM map. In contrast, RDF structures deposited in the PDB can represent conformations that not only differ from the consensus obtained by flexible fitting but also from X-ray crystallography. Thus, this study emphasizes that a "consensus" achieved by the use of several automated flexible fitting approaches can provide a higher level of confidence in the modeled configurations. Following this protocol not only increases the confidence level of fitting, but also highlights protein regions with uncertain fitting. Hence, this protocol can lead to better interpretation of cryo-EM data. 相似文献
20.
Jun Zhu Pawel A. Penczek Rasmus Schröder Joachim Frank 《Journal of structural biology》1997,118(3):197-219
Cryoelectron microscopy provides the means of studying macromolecules in their native state. However, the contrast transfer function (CTF) makes the images and the three-dimensional (3D) maps derived from them difficult to interpret. We developed methods to determine the CTF from experimental data and to obtain a CTF-corrected 3D reconstruction. The CTF correction and 3D reconstruction accomplished in one step make it easy to combine different defocus data sets and decrease the error accumulation in the computation. These methods were applied to energy-filtered images of the 70SEscherichia coliribosome, resulting in a distortion-free 3D map of the ribosome at 1/24.5 Å−1resolution, as determined by the differential phase residual resolution criterion. 相似文献