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Wah Chiu

Publications and source records attributed to Wah Chiu.

53 records · Page 3Linked to original sources
PubMed2003

Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP.

The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP synthase (F(0)F(1)) that works in close synchrony with the P(i) and ADP/ATP carriers. Each carrier consists of only a single polypeptide chain in dimeric form, while the ATP synthase is highly complex consisting in animals of 17 known subunit types and more than 30 total subunits. Although structures at high resolution have been obtained for the water-soluble F(1) part of the ATP synthase consisting of only five subunit types, such structures have not been obtained for either the complete ATP synthase or the P(i) and ADP/ATP carriers. Here, we report that all three proteins are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4 nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single soluble complexes or "ATP synthasomes" that contain the ATP synthase in association with the P(i) and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed to visualize in atomic detail the entire complex involved. They also provide evidence that the cristae are a subcompartment of the inner membrane.

Animals↗
PubMed2003

Studying large viruses.

In this article we have attempted to describe some structural aspects of large viruses. Although this may seem a straightforward task, it is complicated by the fact that large viruses do not represent a distinctive class of organisms and any grouping under this heading will include a range of unrelated viruses with different structures, replication strategies, and host types. To simplify matters we limited our definition to dsDNA viruses with genomes of 100 kbp or larger. However, even this restricted grouping includes viruses with diverse and seemingly unrelated structures. Furthermore, few if any structural features are exclusive to large viruses and most of what appears distinctive about their structure or assembly can also be found in smaller, and usually better characterized, viruses. Therefore we have not attempted to provide a comprehensive catalog of the properties of large viruses but have tried to illustrate particular structural points with examples from a few of the better known forms, notably herpes simplex virus (HSV) and phage T4. The two techniques used to provide rigorous analyses of virus structures are X-ray crystallography and electron cryomicroscopy with computer-assisted reconstruction. To date, X-ray crystallography has been successful only with smaller viruses, and what is known about the structures of these large viruses has come primarily from electron cryomicroscopy. However, with the notable exception of the HSV capsid, such studies have been limited in extent and of relatively low resolution, and the information obtained has been confined largely to describing the spatial distributions and relationships between the subunits. Nevertheless, these studies have given us our clearest insights into the biology of these complex particles and increases in resolution promise to extend these insights by bridging the gap between gross and atomic structures, as exemplified by the identification and mapping of secondary structural elements in the HSV capsid.

Genome, Viral↗
PubMed2003

Electron beam coater for reduction of charging in ice-embedded biological specimens using Ti(88)Si(12) alloy.

Biological macromolecules embedded in vitreous ice are known to suffer from charging while being imaged in an electron transmission cryomicroscope. We developed an electron beam coater that deposits conductive films onto the surface of frozen-hydrated specimens. The conductive films help to dissipate charge during electron irradiation of poorly conductive ice-embedded biological samples. We observed significant reduction in charging of ice-embedded catalase crystals suspended over holes in a holey carbon film after coating them with a 30-A-thick layer of an amorphous alloy, Ti(88)Si(12). Images of the crystals after coating showed diffraction spots of up to 3 A resolution.

Alloys↗
PubMed2003

Object oriented database and electronic notebook for transmission electron microscopy.

As high-resolution biological transmission electron microscopy (TEM) has increased in popularity over recent years, the volume of data and number of projects underway has risen dramatically. A robust tool for effective data management is essential to efficiently process large data sets and extract maximum information from the available data. We present the Electron Microscopy Electronic Notebook (EMEN), a portable, object-oriented, web-based tool for TEM data archival and project management. EMEN has several unique features. First, the database is logically organized and annotated so multiple collaborators at different geographical locations can easily access and interpret the data without assistance. Second, the database was designed to provide flexibility to the user, so it can be used much as a lab notebook would be, while maintaining a structure suitable for data mining and direct interaction with data-processing software. Finally, as an object-oriented database, the database structure is dynamic and can be easily extended to incorporate information not defined in the original database specification.

Databases, Factual↗
PubMed2003

Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions.

Bacteriophage P22 is a prototypical biological machine used for studying protein complex assembly and capsid maturation. Using cryo-EM, we solved the structures of P22 before and after the capsid maturation at 8.5 A and 9.5 A resolutions, respectively. These structures allowed visualization of alpha-helices and beta-sheets from which the capsid protein fold is derived. The capsid fold is similar to that of the coat protein of HK97 bacteriophage. The cryo-EM shows that a large conformational change of the P22 capsid during maturation transition involves not only the domain movement of individual subunits, but also refolding of the capsid protein.

Bacteriophage P22↗
PubMed2003

Structure of isolated nucleocapsids from venezuelan equine encephalitis virus and implications for assembly and disassembly of enveloped virus.

Venezuelan equine encephalitis virus (VEEV) is an important human and equine pathogen in the Americas, with widespread reoccurring epidemics extending from South America to the southern United States. Most troubling, VEEV has been made into a weapon by several countries and is currently restricted by the Centers for Disease Control and Prevention as a potential biological warfare and terrorism agent. To facilitate the development of antiviral compounds, the structure of the nucleocapsid isolated from VEEV has been determined by electron cryomicroscopy and image reconstruction and represents the first three-dimensional structure of a nucleocapsid isolated from a single-stranded enveloped RNA virus. The isolated VEEV nucleocapsid undergoes significant reorganization relative to its structure within VEEV. However, the isolated nucleocapsid clearly exhibits T=4 icosahedral symmetry, and its characteristic nucleocapsid hexons and pentons are preserved. The diameter of the isolated nucleocapsid is approximately 11.5% larger than that of the nucleocapsid within VEEV, with radial expansion being greatest near the hexons. Significantly, this is the first direct structural evidence showing that a simple enveloped virus undergoes large conformational changes during maturation, suggesting that the lipid bilayer and the transmembrane proteins of simple enveloped viruses provide the energy necessary to reorganize the nucleocapsid during maturation.

Encephalitis Virus, Venezuelan Equine↗
PubMed2002

The skeletal muscle Ca2+ release channel has an oxidoreductase-like domain.

We used a combination of bioinformatics, electron cryomicroscopy, and biochemical techniques to identify an oxidoreductase-like domain in the skeletal muscle Ca2+ release channel protein (RyR1). The initial prediction was derived from sequence-based fold recognition for the N-terminal region (41-420) of RyR1. The putative domain was computationally localized to the clamp domain in the cytoplasmic region of a 22A structure of RyR1. This localization was subsequently confirmed by difference imaging with a sequence specific antibody. Consistent with the prediction of an oxidoreductase domain, RyR1 binds [3H]NAD+, supporting a model in which RyR1 has a oxidoreductase-like domain that could function as a type of redox sensor.

Amino Acid Sequence↗
PubMed2002

Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum.

VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.

Adenosine Triphosphatases↗
PubMed2002

High resolution structural studies of complex icosahedral viruses: a brief overview.

Structural descriptions of viral particles are key to our understanding of their assembly mechanisms and properties. We will describe the application of X-ray crystallography and electron cryomicroscopy to the structural determination of the bluetongue virus core and the herpesvirus capsid. These represent the highest resolution structural studies carried out by these techniques on such complex and large icosahedral virus particles. The bluetongue virus core consists of two layers of distinct proteins with different protein packing symmetries, while the herpes virus capsid is made up of four types of proteins with 3.3 MDa per asymmetric unit. The structural results reveal that each of these proteins has distinct folds and they are packed uniquely to form stable particles.

Animals↗
PubMed2002

Bilamellar cationic liposomes protect adenovectors from preexisting humoral immune responses.

Adenoviral vectors have been widely used for gene therapy, but they are limited both by the presence of a humoral immune response that dramatically decreases the level of transduction after reinjection and by their requirement for target cells to express appropriate receptors such as Coxsackie adenovirus receptor (CAR). To overcome both limits, we encapsulated adenovectors using bilamellar DOTAP:chol liposomes. Electron micrography (EM) showed that these liposomes efficiently encapsulated the vectors, allowing CAR-independent adenovector transduction of otherwise resistant cells. DOTAP:chol-encapsulated adenovectors encoding LacZ or alpha(1)-antitrypsin inhibitor (AAT) were also functionally resistant ex vivo and in vivo to the neutralizing effects of human anti-adenoviral antibodies, unlike other liposomal systems. Hence, bilamellar DOTAP:chol liposomes may be useful for applications using adenovectors in which the target cells lack adenoviral receptors or in which the recipient already has or develops a neutralizing antibody response that would otherwise inactivate readministered vector.

Adenoviridae↗
PubMed2002

Deriving folds of macromolecular complexes through electron cryomicroscopy and bioinformatics approaches.

Intermediate-resolution (7-9A) structures of large macromolecular complexes can be obtained by electron cryomicroscopy. This structural information, combined with bioinformatics data for the individual protein components or domains, can lead to a fold model for the entire complex. Such approaches have been demonstrated with the 6.8 A structure of the rice dwarf virus to derive models for the major capsid shell proteins.

Amino Acid Sequence↗
PubMed2001

Quaternary structure of human fatty acid synthase by electron cryomicroscopy.

We present the first three-dimensional reconstruction of human fatty acid synthase obtained by electron cryomicroscopy and single-particle image processing. The structure shows that the synthase is composed of two monomers, arranged in an antiparallel orientation, which is consistent with biochemical data. The monomers are connected to each other at their middle by a bridge of density, a site proposed to be the combination of the interdomain regions of the two monomers. Each monomer subunit appears to be subdivided into three structural domains. With this reconstruction of the synthase, we propose a location for the enzyme's two fatty acid synthesis sites.

Binding Sites↗
PubMed2001

Web-based Simulation for Contrast Transfer Function and Envelope Functions.

We have implemented a Web-based simulation program for the contrast transfer function and envelope functions in Java and JavaScript. The simulation provides interactive controls of all the parameters in those functions. In addition to the predefined functions, users can easily define new functions that use any of the parameters in the preset functions. The most useful feature of this new simulation program is the convenient, universal accessibility through Web browsers on any computer platform that supports Java, such as Netscape and Internet Explorer.

Journal Article↗
PubMedDate not supplied

Applications of a bilateral denoising filter in biological electron microscopy.

Due to the sensitivity of biological sample to the radiation damage, the low dose imaging conditions used for electron microscopy result in extremely noisy images. The processes of digitization, image alignment, and 3D reconstruction also introduce additional sources of noise in the final 3D structure. In this paper, we investigate the effectiveness of a bilateral denoising filter in various biological electron microscopy applications. In contrast to the conventional low pass filters, which inevitably smooth out both noise and structural features simultaneously, we found that bilateral filter holds a distinct advantage in being capable of effectively suppressing noise without blurring the high resolution details. In as much, we have applied this technique to individual micrographs, entire 3D reconstructions, segmented proteins, and tomographic reconstructions.

Anisotropy↗
PubMedDate not supplied

Focal pair merging for contrast enhancement of single particles.

A common technique in transmission electron microscopy is the collection of a focal pair, in which the first, close to focus image contains higher resolution information at lower contrast, and the second, far from focus image has high contrast but less reliable high-resolution information. Typically these second micrographs are used for purposes of particle selection, orientation estimate or micrograph evaluation. We introduce a technique for merging the information from both images, including signal to noise ratio weighting, contrast transfer function correction, and optional Weiner filtration. This produces a composite image with reduced contrast transfer function artifacts and optimized contrast. This technique is useful in numerous cases where low-contrast images are produced, such as small particles, proteins solubilized in detergent or projects with high-resolution goals when the first image is taken very close to focus.

Algorithms↗