An ab initio algorithm for low-resolution 3-D reconstructions from cryoelectron microscopy images

A statistical method for determining low-resolution 3-D reconstructions of virus particles from cryoelectron microscope images by an ab initio algorithm is described. The method begins with a novel linear reconstruction method that generates a spherically symmetric reconstruction, which is followed by a nonlinear reconstruction method implementing an expectation-maximization procedure using the spherically symmetric reconstruction as an initial condition and resulting in a reconstruction with icosahedral symmetry. An important characteristic of the complete method is that very little need be known about the particle before the reconstruction is computed, in particular, only the type of symmetry and inner and outer radii. The method is demonstrated on synthetic cowpea mosaic virus data, and its robustness to 5% errors in the contrast transfer function, 5% errors in the location of the center of the particles in the images, and 5% distortion in the 3-D structure from which the images are derived is demonstrated numerically.     

A microcomputer based system for three-dimensional reconstructions from tomographic or histologic sections

The reconstructions of three-dimensional (3-D) objects from serial two-dimensional (2-D) images can contribute to the understanding of many biologic structures, from organelles to organs and tissues. The 3-D reconstruction of sections can be divided into several major tasks: image acquisition, alignment of slices, internal object definition, object reconstruction and rotation of the completed image. A fast, versatile, interactive system was devised for the reconstruction of 3-D objects from serial 2-D images using a low-cost microcomputer, original programs and commercial software. The system allows reconstruction from any serial images, e.g., electron micrographs, histologic sections or computed tomograms. A photographic image or a microscopic field is acquired into the computer memory using a video digitizer. Slices are superimposed and aligned to each other using an operator-interactive program. A contour-(edge-) finding algorithm isolates an object of interest from the background image by "subtraction" of the image from an overlaid, slightly shifted identical image. Contours for each slice are input to a reconstruction procedure, which calculates the x, y and z coordinates of every point in a slice and the thickness and number of slices. It then calculates the illumination for every point using a given point source of light and an intensity-fading coefficient. Finally, the points are represented by cubes to provide dimension and reflective surfaces. A cube of appropriate shade and color represents in 2-D the equivalent of a 3-D object; this results in a very effective 3-D image. The reconstruction is rotated by recalculating the positions of every point defining the object and rebuilding the image.(ABSTRACT TRUNCATED AT 250 WORDS)     

SwarmPS: rapid, semi-automated single particle selection software

Single particle analysis (SPA) coupled with high-resolution electron cryo-microscopy is emerging as a powerful technique for the structure determination of membrane protein complexes and soluble macromolecular assemblies. Current estimates suggest that approximately 10(4)-10(5) particle projections are required to attain a 3A resolution 3D reconstruction (symmetry dependent). Selecting this number of molecular projections differing in size, shape and symmetry is a rate-limiting step for the automation of 3D image reconstruction. Here, we present Swarm(PS), a feature rich GUI based software package to manage large scale, semi-automated particle picking projects. The software provides cross-correlation and edge-detection algorithms. Algorithm-specific parameters are transparently and automatically determined through user interaction with the image, rather than by trial and error. Other features include multiple image handling (approximately 10(2)), local and global particle selection options, interactive image freezing, automatic particle centering, and full manual override to correct false positives and negatives. Swarm(PS) is user friendly, flexible, extensible, fast, and capable of exporting boxed out projection images, or particle coordinates, compatible with downstream image processing suites.     

Cryo-electron microscope image denoising based on the geodesic distance

Background

To perform a three-dimensional (3-D) reconstruction of electron cryomicroscopy (cryo-EM) images of viruses, it is necessary to determine the similarity of image blocks of the two-dimensional (2-D) projections of the virus. The projections containing high resolution information are typically very noisy. Instead of the traditional Euler metric, this paper proposes a new method, based on the geodesic metric, to measure the similarity of blocks.

Results

Our method is a 2-D image denoising approach. A data set of 2243 cytoplasmic polyhedrosis virus (CPV) capsid particle images in different orientations was used to test the proposed method. Relative to Block-matching and three-dimensional filtering (BM3D), Stein’s unbiased risk estimator (SURE), Bayes shrink and K-means singular value decomposition (K-SVD), the experimental results show that the proposed method can achieve a peak signal-to-noise ratio (PSNR) of 45.65. The method can remove the noise from the cryo-EM image and improve the accuracy of particle picking.

Conclusions

The main contribution of the proposed model is to apply the geodesic distance to measure the similarity of image blocks. We conclude that manifold learning methods can effectively eliminate the noise of the cryo-EM image and improve the accuracy of particle picking.

     

An ab Initio Algorithm for Low-Resolution 3-D Reconstructions from Cryoelectron Microscopy Images

A statistical method for determining low-resolution 3-D reconstructions of virus particles from cryoelectron microscope images by an ab initio algorithm is described. The method begins with a novel linear reconstruction method that generates a spherically symmetric reconstruction, which is followed by a nonlinear reconstruction method implementing an expectation-maximization procedure using the spherically symmetric reconstruction as an initial condition and resulting in a reconstruction with icosahedral symmetry. An important characteristic of the complete method is that very little need be known about the particle before the reconstruction is computed, in particular, only the type of symmetry and inner and outer radii. The method is demonstrated on synthetic cowpea mosaic virus data, and its robustness to 5% errors in the contrast transfer function, 5% errors in the location of the center of the particles in the images, and 5% distortion in the 3-D structure from which the images are derived is demonstrated numerically.     

The three-dimensional structure of frozen-hydrated Nudaurelia capensis β Virus, a T = 4 insect Virus

The three-dimensional structure of Nudaurelia capensis beta virus (N beta V) was reconstructed to 3.2-nm resolution from images of frozen-hydrated virions. The distinctly icosahedral capsid (approximately 40-nm diameter) contains 240 copies of a single 61-kDa protein subunit arranged with T = 4 lattice symmetry. The outer surface of unstained virions compares remarkably well with that previously observed in negatively stained specimens. Inspection of the density map, volume estimates, and model building experiments indicate that each subunit consists of two distinct domains. The large domain (approximately 40 kDa) has a cylindrical shape, approximately 4-nm diameter by approximately 4-nm high, and associates with two large domains of neighboring subunits to form a Y-shaped trimeric aggregate in the outer capsid surface. Four trimers make up each of the 20 planar faces of the capsid. Small domains (approximately 21 kDa) presumably associate at lower radii (approximately 13-16.5 nm) to form a contiguous, non-spherical shell. A T = 4 model, constructed from 80 trimers of the common beta-barrel core motif (approximately 20 kDa) found in many of the smaller T = 3 and pseudo T = 3 viruses, fits the dimensions and features seen in the N beta V reconstruction, suggesting that the contiguous shell of N beta V may be formed by intersubunit contacts between small domains having that motif. The small (approximately 1800 kDa), ssRNA genome is loosely packed inside the capsid with a low average density.     

Three-dimensional structure of the Chinese Sacbrood bee virus

The RNA of Chinese Sacbrood Bee Virus (CSBV) was purified and used as template to obtain a 1096 bp cDNA fragment by RT-PCR amplification. This DNA fragment was cloned into pGEM-T Easy Vector for sequencing. Analyses of the sequenced CSBV RNA fragment revealed a nucleotide sequence homology of 87.6% and a deduced amino-acid sequence homology of 94.6% with that of the Sacbrood Virus (SBV), indicating that CSBV is a different but highly homologous virus of SBV. The three-dimensional (3D) structure of CSBV was determined at 2.5 nm resolution by using electron cryo-microscopy (cryoEM) and computer reconstruction methods. The 3-D structure showed that the capsid has aT = 1 (orP = 3) icosahedral capsid shell with a smooth surface. There were 12 pentons at its icosahedral vertices (5-fold axes) and 132 holes penetrating the shell. The 3-D structure also revealed densities corresponding to the CSBV genome, suggesting icosahedrally-ordered RNA organization, a novel feature not previously reported for any picornaviruses.     

Three-dimensional structure of the Chinese Sacbrood bee virus

The RNA of Chinese Sacbrood Bee Virus (CSBV) was purified and used as template to obtain a 1096 bp cDNA fragment by RT-PCR amplification. This DNA fragment was cloned into pGEM-T Easy Vector for sequencing. Analyses of the sequenced CSBV RNA fragment revealed a nucleotide sequence homology of 87.6% and a deduced amino-acid sequence homology of 94.6% with that of the Sacbrood Virus (SBV), indicating that CSBV is a different but highly homologous virus of SBV. The three-dimensional (3D) structure of CSBV was determined at 2.5 nm resolution by using electron cryo-microscopy (cryoEM) and computer reconstruction methods. The 3-D structure showed that the capsid has a T= 1 (or P= 3) icosahedral capsid shell with a smooth surface. There were 12 pentons at its icosahedral vertices (5-fold axes) and 132 holes penetrating the shell. The 3-D structure also revealed densities corresponding to the CSBV genome, suggesting icosahedrally-ordered RNA organization, a novel feature not previously reported for any picornavi     

The marine algal virus PpV01 has an icosahedral capsid with T=219 quasisymmetry

Phaeocystis pouchetii virus (PpV01) infects and lyses the haptophyte Phaeocystis pouchetii (Hariot) Lagerheim and was first isolated from Norwegian coastal waters. We have used electron cryomicroscopy and three-dimensional image reconstruction methods to examine the native morphology of PpV01 at a resolution of 3 nm. The icosahedral capsid of PpV01 has a maximum diameter of 220 nm and is composed of 2,192 capsomers arranged with T=219 quasisymmetry. One specific capsomer in each asymmetric unit contains a fiber-like protrusion. Density attributed to the presence of a lipid membrane appears just below (inside) the capsid. PpV01 is the largest icosahedral virus whose capsid structure has been determined in three dimensions from images of vitrified samples. Striking similarities in the structures of PpV01 and a number of other large double-stranded DNA viruses are consistent with a growing body of evidence that they share a common evolutionary origin.     

Dissecting quasi-equivalence in nonenveloped viruses: membrane disruption is promoted by lytic peptides released from subunit pentamers, not hexamers

Nonenveloped viruses often invade membranes by exposing hydrophobic or amphipathic peptides generated by a proteolytic maturation step that leaves a lytic peptide noncovalently associated with the viral capsid. Since multiple copies of the same protein form many nonenveloped virus capsids, it is unclear if lytic peptides derived from subunits occupying different positions in a quasi-equivalent icosahedral capsid play different roles in host infection. We addressed this question with Nudaurelia capensis omega virus (NωV), an insect RNA virus with an icosahedral capsid formed by protein α, which undergoes autocleavage during maturation, producing the lytic γ peptide. NωV is a unique model because autocatalysis can be precisely initiated in vitro and is sufficiently slow to correlate lytic activity with γ peptide production. Using liposome-based assays, we observed that autocatalysis is essential for the potent membrane disruption caused by NωV. We observed that lytic activity is acquired rapidly during the maturation program, reaching 100% activity with less than 50% of the subunits cleaved. Previous time-resolved structural studies of partially mature NωV particles showed that, during this time frame, γ peptides derived from the pentamer subunits are produced and are organized in a vertical helical bundle that is projected toward the particle surface, while identical polypeptides in quasi-equivalent subunits are produced later or are in positions inappropriate for release. Our functional data provide experimental support for the hypothesis that pentamers containing a central helical bundle, observed in different nonenveloped virus families, are a specialized lytic motif.     

Pattern formation in icosahedral virus capsids: the papova viruses and Nudaurelia capensis beta virus.

The capsids of the spherical viruses all show underlying icosahedral symmetry, yet they differ markedly in capsomere shape and in capsomere position and orientation. The capsid patterns presented by the capsomere shapes, positions, and orientations of three viruses (papilloma, SV40, and N beta V) have been generated dynamically through a bottom-up procedure which provides a basis for understanding the patterns. A capsomere shape is represented in two-dimensional cross-section by a mass or charge density on the surface of a sphere, given by an expansion in spherical harmonics, and referred to herein as a morphological unit (MU). A capsid pattern is represented by an icosahedrally symmetrical superposition of such densities, determined by the positions and orientations of its MUs on the spherical surface. The fitness of an arrangement of MUs is measured by an interaction integral through which all capsid elements interact with each other via an arbitrary function of distance. A capsid pattern is generated by allowing the correct number of approximately shaped MUs to move dynamically on the sphere, positioning themselves until an extremum of the fitness function is attained. The resulting patterns are largely independent of the details of both the capsomere representation and the interaction function; thus the patterns produced are generic. The simplest useful fitness function is sigma 2, the average square of the mass (or charge) density, a minimum of which corresponds to a "uniformly spaced" MU distribution; to good approximation, the electrostatic free energy of charged capsomeres, calculated from the linearized Poisson-Boltzmann equation, is proportional to sigma 2. With disks as MUs, the model generates the coordinated lattices familiar from the quasi-equivalence theory, indexed by triangulation numbers. Using fivefold MUs, the model generates the patterns observed at different radii within the T = 7 capsid of papilloma and at the surface of SV40; threefold MUs give the T = 4 pattern of Nudaurelia capensis beta virus. In all cases examined so far, the MU orientations are correctly found.     

Auto-accumulation method using simulated annealing enables fully automatic particle pickup completely free from a matching template or learning data

Single-particle analysis is a 3-D structure determining method using electron microscopy (EM). In this method, a large number of projections is required to create 3-D reconstruction. In order to enable completely automatic pickup without a matching template or a training data set, we established a brand-new method in which the frames to pickup particles are randomly shifted and rotated over the electron micrograph and, using the total average image of the framed images as an index, each frame reaches a particle. In this process, shifts are selected to increase the contrast of the average. By iterated shifts and further selection of the shifts, the frames are induced to shift so as to surround particles. In this algorithm, hundreds of frames are initially distributed randomly over the electron micrograph in which multi-particle images are dispersed. Starting with these frames, one of them is selected and shifted randomly, and acceptance or non-acceptance of its new position is judged using the simulated annealing (SA) method in which the contrast score of the total average image is adopted as an index. After iteration of this process, the position of each frame converges so as to surround a particle and the framed images are picked up. This method is the first unsupervised fully automatic particle picking method which is applicable to EM of various kinds of proteins, especially to low-contrasted cryo-EM protein images.     

Real space refinement of acto-myosin structures from sectioned muscle

We have adapted a real space refinement protocol originally developed for high-resolution crystallographic analysis for use in fitting atomic models of actin filaments and myosin subfragment 1 (S1) to 3-D images of thin-sectioned, plastic-embedded whole muscle. The rationale for this effort is to obtain a refinement protocol that will optimize the fit of the model to the density obtained by electron microscopy and correct for poor geometry introduced during the manual fitting of a high-resolution atomic model into a lower resolution 3-D image. The starting atomic model consisted of a rigor acto-S1 model obtained by X-ray crystallography and helical reconstruction of electron micrographs. This model was rebuilt to fit 3-D images of rigor insect flight muscle at a resolution of 7 nm obtained by electron tomography and image averaging. Our highly constrained real space refinement resulted in modest improvements in the agreement of model and reconstruction but reduced the number of conflicting atomic contacts by 70% without loss of fit to the 3-D density. The methodology seems to be well suited to the derivation of stereochemically reasonable atomic models that are consistent with experimentally determined 3-D reconstructions computed from electron micrographs.     

基于VTK的医学图像三维可视化系统

医学图像的三维可视化可以通过可视化工具包（VTK）提供的API实现。VTK是医学图像可视化的开法工具包,它把可视化的算法封装起来,利用简单的代码生成所需图形。基于VTK的医学图像三维可视化系统阐述了如何借助VTKAPI读入二维医学图像序列、操作二维图像、重建三维图像以及进行三维图像可视化的全套方案,为临床医生的诊断、治疗提供了有益的途径。     

An ImageJ-based tool for three-dimensional registration between different types of microscopic images

Three-dimensional (3D) registration (i.e., alignment) between two microscopic images is very helpful to study tissues that do not adhere to substrates, such as mouse embryos and organoids, which are often 3D rotated during imaging. However, there is no 3D registration tool easily accessible for experimental biologists. Here we developed an ImageJ-based tool which allows for 3D registration accompanied with both quantitative evaluation of the accuracy and reconstruction of 3D rotated images. In this tool, several landmarks are manually provided in two images to be aligned, and 3D rotation is computed so that the distances between the paired landmarks from the two images are minimized. By simultaneously providing multiple points (e.g., all nuclei in the regions of interest) other than the landmarks in the two images, the correspondence of each point between the two images, i.e., to which nucleus in one image a certain nucleus in another image corresponds, is quantitatively explored. Furthermore, 3D rotation is applied to one of the two images, resulting in reconstruction of 3D rotated images. We demonstrated that this tool successfully achieved 3D registration and reconstruction of images in mouse pre- and post-implantation embryos, where one image was obtained during live imaging and another image was obtained from fixed embryos after live imaging. This approach provides a versatile tool applicable for various tissues and species.     

4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells

Background

Synthetic biology is a discipline that includes making life forms artificially from chemicals. Here, a DNA molecule was enzymatically synthesized in vitro from DNA templates made from oligonucleotides representing the text of the first Tobacco mosaic virus (TMV) sequence elucidated in 1982. No infectious DNA molecule of that seminal reference sequence exists, so the goal was to synthesize it and then build viral chimeras.

Results

RNA was transcribed from synthetic DNA and encapsidated with capsid protein in vitro to make synthetic virions. Plants inoculated with the virions did not develop symptoms. When two nucleotide mutations present in the original sequence, but not present in most other TMV sequences in GenBank, were altered to reflect the consensus, the derivative synthetic virions produced classic TMV symptoms. Chimeras were then made by exchanging TMV capsid protein DNA with Tomato mosaic virus (ToMV) and Barley stripe mosaic virus (BSMV) capsid protein DNA. Virus expressing ToMV capsid protein exhibited altered, ToMV-like symptoms in Nicotiana sylvestris. A hybrid ORF6 protein unknown to nature, created by substituting the capsid protein genes in the virus, was found to be a major symptom determinant in Nicotiana benthamiana. Virus expressing BSMV capsid protein did not have an extended host range to barley, but did produce novel symptoms in N. benthamiana.

Conclusions

This first report of the chemical synthesis and artificial assembly of a plant virus corrects a long-standing error in the TMV reference genome sequence and reveals that unnatural hybrid virus proteins can alter symptoms unexpectedly.     

Molecular Packing in Virus Crystals: Geometry, Chemistry, and Biology

An automated procedure was developed to determine the geometrical and chemical interactions of crystalline virus particles using the crystal parameters, particle position, orientation, and atomic coordinates for an icosahedral asymmetric unit. Two applications of the program are reported: (1) An analysis of a novelpseudo-rhombohedral (R32) symmetry present in the monoclinic crystal lattices of both Nodamura Virus (NOV) and Coxsackie virus B3 (CVB3). The study shows that in both cases the interactions between particles is substantially increased by minor deviations from exact R32 symmetry and that only particles with the proper ratio of dimensions along twofold and fivefold symmetry axes (such as southern bean mosaic virus) can achieve comparable buried surface area in the true R32 space group. (2) An attempt was made to correlate biological function with remarkably conserved interparticle contact regions found in different crystal forms of three members of the nodavirus family, NOV, Flock House Virus (FHV), and Black Beetle Virus (BBV). Mutational evidence implicates the quasi-threefold region on the viral surface in receptor binding in nodaviruses and this region is dominant in particle contacts in all three virus crystals. Examination of particle contacts in numerous crystal structures of viruses in the picornavirus superfamily showed that portions of the capsid surface known to interact with a receptor or serve as an epitope for monoclonal antibodies frequently stabilize crystal contacts.     

Dynamics and Stability in Maturation of a T = 4 Virus

Nudaurelia capensis ω virus is a T = 4, icosahedral virus with a bipartite, positive-sense RNA genome. Expression of the coat protein gene in a baculovirus system was previously shown to result in the formation of procapsids when purified at pH 7.6. Procapsids are round, porous particles (480 Å diameter) and have T = 4 quasi-symmetry. Reduction of pH from 7.6 to 5.0 resulted in virus-like particles (VLP_5.0) that are morphologically identical with authentic virions, with an icosahedral-shaped capsid and a maximum dimension of 410 Å. VLP_5.0 undergoes a maturation cleavage between residues N570 and F571, creating the covalently independent γ peptide (residues 571-641) that remains associated with the particle. This cleavage also occurs in authentic virions, and in each case, it renders the morphological change irreversible (i.e., capsids do not expand when the pH is raised back to 7.6). However, a non-cleavable mutant, N570T, undergoes the transition reversibly (NT_7.6 ↔ NT_5.0). We used electron cryo-microscopy and three-dimensional image reconstruction to study the icosahedral structures of NT_7.6, NT_5.0, and VLP_5.0 at about 8, 6, and 6 Å resolution, respectively. We employed the 2. 8-Å X-ray model of the mature virus, determined at pH 7.0 (XR_7.0), to establish (1) how and why procapsid and capsid structures differ, (2) why lowering pH drives the transition, and (3) why the non-cleaving NT_5.0 is reversible. We show that procapsid assembly minimizes the differences in quaternary interactions in the particle. The two classes of 2-fold contacts in the T = 4 surface lattice are virtually identical, both mediated by similarly positioned but dynamic γ peptides. Furthermore, quasi and icosahedral 3-fold interactions are indistinguishable. Maturation results from neutralizing the repulsive negative charge at subunit interfaces with significant differentiation of quaternary interactions (one 2-fold becomes flat, mediated by a γ peptide, while the other is bent with the γ peptide disordered) and dramatic stabilization of the particle. The γ peptide at the flat contact remains dynamic when cleavage cannot occur (NT_5.0) but becomes totally immobilized by noncovalent interactions after cleavage (VLP_5.0).