AVS Software for Visualization in Molecular Microscopy

Sheehan, Brian; Fuller, Stephen D.; Pique, Michael E.; Yeager, Mark

doi:10.1006/jsbi.1996.0017

Cited by 41 publications

(23 citation statements)

References 23 publications

(24 reference statements)

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“…This was a conservative estimate, since the final map was computed from twice as many particles. All surface-shaded representations were visualized using AVS software (19). Contour levels were chosen to include the volume occupied by the capsid shell calculated from the number of the copies and the molecular weight of the coat protein.…”

Section: Methodsmentioning

confidence: 99%

Nodavirus Coat Protein Imposes Dodecahedral RNA Structure Independent of Nucleotide Sequence and Length

Tihova¹,

Dryden²,

et al. 2004

J Virol

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The nodavirus Flock house virus (FHV) has a bipartite, positive-sense RNA genome that is packaged into an icosahedral particle displaying T‫3؍‬ symmetry. The high-resolution X-ray structure of FHV has shown that 10 bp of well-ordered, double-stranded RNA are located at each of the 30 twofold axes of the virion, but it is not known which portions of the genome form these duplex regions. The regular distribution of double-stranded RNA in the interior of the virus particle indicates that large regions of the encapsidated genome are engaged in secondary structure interactions. Moreover, the RNA is restricted to a topology that is unlikely to exist during translation or replication. We used electron cryomicroscopy and image reconstruction to determine the structure of four types of FHV particles that differed in RNA and protein content. RNA-capsid interactions were primarily mediated via the N and C termini, which are essential for RNA recognition and particle assembly. A substantial fraction of the packaged nucleic acid, either viral or heterologous, was organized as a dodecahedral cage of duplex RNA. The similarity in tertiary structure suggests that RNA folding is independent of sequence and length. Computational modeling indicated that RNA duplex formation involves both short-range and long-range interactions. We propose that the capsid protein is able to exploit the plasticity of the RNA secondary structures, capturing those that are compatible with the geometry of the dodecahedral cage.High-resolution X-ray analysis of icosahedral viruses has provided detailed insights into the organization of the viral capsid and the structure of the individual coat protein subunits. The encapsidated nucleic acid, on the other hand, is rarely visualized, in part because it does not conform to the icosahedral symmetry of the virus particle. There are exceptions to this, however, as exemplified by the plant viruses bean pod mottle virus (BPMV) (3) and satellite tobacco mosaic virus (STMV) (10) and the invertebrate nodaviruses Pariacoto virus (PaV) (20) and Flock house virus (FHV) (7). In these viruses, regions of the encapsidated single-stranded RNA genome interact with coat protein subunits at symmetrically equivalent positions, and an average structure of these ordered regions has been visualized at high resolution.BPMV, a Tϭ3 icosahedral virus, has a bipartite positivesense single-stranded RNA genome, with each RNA molecule packaged in a separate particle. In the crystal structure of particles containing RNA2 (ϳ3.6 kb), seven well-ordered ribonucleotides were visible near the icosahedral threefold axes of the virion (3). In addition, a total of 660 ribonucleotides corresponding to almost 20% of the packaged RNA could be modeled into the electron density. The overall structure of the ordered RNA is a single-stranded helix, which approximates that found for one strand of an A-type RNA duplex (3, 4). Because the RNA density in the X-ray map represents an average of the densities at symmetrically equivalent positions, the nucleot...

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Section: Methodsmentioning

confidence: 99%

Nodavirus Coat Protein Imposes Dodecahedral RNA Structure Independent of Nucleotide Sequence and Length

Tihova¹,

Dryden²,

et al. 2004

J Virol

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“…Particle images were corrected for the effect of the contrast transfer function (CTF) by phase reversal (20). Surface-rendered views of the final 3D map were visualized by using AVS software (21).…”

mentioning

confidence: 99%

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis

Fonseca¹,

Morris²,

Nerou³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Materials and Methods Purification of IP3R.A membrane fraction was prepared from rat cerebella (17), resuspended (50 mg͞ml in 50 mM Tris͞1 mM EDTA, pH 8.3), and solubilized by the addition of 1% Triton X-100. After centrifugation (100,000 ϫ g for 60 min), the supernatant, supplemented with 250 mM NaCl, was loaded onto an Econopac heparin column (1 ml, Bio-Rad), washed with 25 ml of medium A (50 mM Tris͞250 mM NaCl͞10% glycerol, pH 8.3), supplemented with 1 mM EDTA, 1% Surfact-Amps X-100 (Pierce), and then with medium A supplemented with 0.1% Surfact-Amps X-100 (50 ml). IP 3 R was eluted from the heparin column directly onto a 2-ml Con A-agarose column (Sigma) by using medium A supplemented with 0.1% Surfact-Amps X-100 and 50 M decavanadate (18). After washing with 150 ml of medium B (50 mM Tris͞100 mM NaCl͞10% glycerol͞0.1% Surfact-Amps X-100, pH 8.3), IP 3 R was eluted by overnight incubation with 4 ml of medium B containing 800 mM methyl mannopyranoside (17). To assess the quaternary structure of the purified receptor, 0.2-ml fractions of the final eluate were loaded onto linear 10-30% sucrose gradients (10 ml) in 50 mM Tris͞1 mM EDTA͞0.1% Surfact-Amps X-100, pH 8.3, and centrifuged (134,000 ϫ g for 60 min). Gradients were calibrated by using a high-molecular-weight gel filtration standards kit (Amersham Biosciences). Size-exclusion HPLC was preformed by using a Zorbax GF-450 column (Jones Chromatography, Lakewood, CO) coupled to a Kontron system (Kontron Instruments, Watford, U.K.). All media included the following mixture of protease inhibitors: 150 nM aprotinin, 1 M pepstatin, 20 g͞ml soybean trypsin inhibitor, 1 mM benzamidine, 250 M PMSF, 250 M captopril, and 1 M bestatin.

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“…For the 3D reconstitution work, we used the software package AVS (Porwol et al 1996;Sheehan et al 1996). This approach is convenient because of the high flexibility provided by the programming environment.…”

Section: Discussionmentioning

confidence: 99%

“…We developed programs on the basis of AVS to perform the data visualization. A brief description of the AVS system with special emphasis on microscopy has recently been published (Sheehan et al 1996). The features were processed as follows.…”

Section: Three-dimensional Reconstructionmentioning

confidence: 99%

“…A high number (60 or 79) of optical sections was obtained from the double-stained translucent assemblies. For the 3D reconstitution of the sectioned space, we used the software package "Application Visualization System" (AVS) (Foley et al 1994;Sheehan et al 1996) that was modified to meet the requirements of confocal laser scanning microscopy (Porwol et al 1996). The reconstructed images obtained show the gel matrix, the cell surface, and the spatial relationship of the stained intracellular compartments containing cathepsin B.…”

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confidence: 99%

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Tomography of Cells by Confocal Laser Scanning Microscopy and Computer-assisted Three-dimensional Image Reconstruction: Localization of Cathepsin B in Tumor Cells Penetrating Collagen Gels In Vitro

Strohmaier

Porwol

Acker

et al. 1997

J Histochem Cytochem.

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SUMMARYWe used the nondestructive procedures of confocal laser scanning microscopy in combination with computer-assisted methods to visualize tumor cells in the process of penetrating collagen gels. Three independent sets of images were collected. The image information of all data sets was combined into one image, giving a three-dimensional (3D) impression at high light microscopic resolution and sensitivity. We collected information about the extracellular matrix using the reflection mode, the cell surface/morphology by staining with the fluorescent dye DiOC 6 (3), and the distribution of cathepsin B by Cy-3-labeled immunolocalization. The specific aim of our study was visualization of the spatial relationship of cell organelles as far as they contain the enzyme cathepsin B to cell morphology and motility in a 3D model of extracellular matrix. The majority of the enzyme was localized pericellularly, with no visible relationship to the direction of movement. However, substantial amounts also appeared in intramatrix pseudopodia and associated with the extracellular face of the plasma membane, which may be indicative either of secretion and/or epicellular activity. Our approach has general applicability to study of the spatial relationships of Biological structures have a dynamic three-dimensional (3D) organization. However, our understanding of structures and functions and their interrelations in cells is almost exclusively based on two-dimensional images and is therefore incomplete. In medical sciences, 3D imagery of whole organs or large anatomic regions of the body is possible by a number of noninvasive methods. Moreover, at the molecular level, spatial reconstitution of molecules or molecular assemblies is a common, although complicated procedure. At the cellular level, knowledge of spatial extension or interaction of structures at the light and electron microscopic level was obtained by destructive sectioning methods. Confocal laser scanning microscopy is now a well-established method to obtain optical sections of spatially extended biological objects (Brakenhoff et al. 1989;Masters and Farmer 1993;Wright et al. 1993). This precise and nondestructive procedure can render series of a sufficient number of equally thick sections to allow accurate 3D reconstitution of the imaged volume. The invention of appropriate fluorescent dyes and the development of specific antibodies to cell constituents are the last links in a chain leading to realistic 3D images of cells and their constituents.Such a methodology is an ideal tool to study cell locomotion (movement). In situ, this is a 3D process. It involves complex rearrangements of the intracellular compartments, intercellular interactions, and interactions of cells with the extracellular matrix. Movement of cells through natural barriers (invasion) is a basic phenomenon in living organisms. In higher organisms, such invasive processes occur during all stages of deCorrespondence to: Dr. Eberhard Spiess, Biomedizinische Strukturforschung 0195,

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AVS Software for Visualization in Molecular Microscopy

Cited by 41 publications

References 23 publications

Nodavirus Coat Protein Imposes Dodecahedral RNA Structure Independent of Nucleotide Sequence and Length

Nodavirus Coat Protein Imposes Dodecahedral RNA Structure Independent of Nucleotide Sequence and Length

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis

Tomography of Cells by Confocal Laser Scanning Microscopy and Computer-assisted Three-dimensional Image Reconstruction: Localization of Cathepsin B in Tumor Cells Penetrating Collagen Gels In Vitro

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