Biomedical subjects

P A Thuman-Commike

Publications and source records attributed to P A Thuman-Commike.

10 recordsLinked to original sources

PubMed2001

Single particle macromolecular structure determination via electron microscopy.

Three-dimensional structure determination of macromolecules and macromolecular complexes is an integral part of understanding biological functions. For large protein and macromolecular complexes structure determination is often performed using electron cryomicroscopy where projection images of individual macromolecular complexes are combined to produce a three-dimensional reconstruction. Single particle methods have been devised to perform this structure determination for macromolecular complexes with little or no underlying symmetry. These computational methods generally involve an iterative process of aligning unique views of the macromolecular images followed by determination of the angular components that define those views. In this review, this structure determination process is described with the aim of clarifying a seemingly complex structural method.

Bacteriophages↗

PubMed2000

Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy.

Large-scale conformational transitions are involved in the life-cycle of many types of virus. The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding-containing precursor capsid to the DNA-containing mature virion. This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons. We have used electron cryomicroscopy and image reconstruction to obtain 15 A structures of both bacteriophage P22 procapsids and mature phage. The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits. The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface. As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation. We also observe DNA within the capsid packed in a manner consistent with the spool model. These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids.

Bacteriophage P22↗

PubMed2000

Reconstruction principles of icosahedral virus structure determination using electron cryomicroscopy.

Electron cryomicroscopy is a useful tool for studying the three-dimensional structure of icosahedral viruses. This review is intended to provide beginners with an understanding of icosahedral virus structure determination focusing on the data processing aspects. We begin with an overview of the entire structure determination process and a brief summary of the sample preparation and imaging aspects. Next, we provide detailed descriptions of each data processing step leading to three-dimensional reconstruction, including application of image corrections, resolution assessment, and structure visualization. To aid in understanding this reconstruction process we provide a variety of illustrative examples. Last, we summarize future prospects for icosahedral virus structural studies.

Bacteriophage lambda↗

PubMed2000

Identification of additional coat-scaffolding interactions in a bacteriophage P22 mutant defective in maturation.

Scaffolding proteins play a critical role in the assembly of certain viruses by directing the formation and maturation of a precursor capsid. Using electron cryomicroscopy difference mapping, we have identified an altered arrangement of a mutant scaffolding within the bacteriophage P22 procapsid. This mutant scaffolding allows us to directly visualize scaffolding density within the P22 procapsid. Based on these observations we propose a model for why the mutant prevents scaffolding release and capsid maturation.

Bacteriophage P22↗

PubMed1999

Solution x-ray scattering-based estimation of electron cryomicroscopy imaging parameters for reconstruction of virus particles.

Structure factor amplitudes and phases can be computed directly from electron cryomicroscopy images. Inherent aberrations of the electromagnetic lenses and other instrumental factors affect the structure factors, however, resulting in decreased accuracy in the determined three-dimensional reconstruction. In contrast, solution x-ray scattering provides absolute and accurate measurement of spherically averaged structure factor amplitudes of particles in solution but does not provide information on the phases. In the present study, we explore the merits of using solution x-ray scattering data to estimate the imaging parameters necessary to make corrections to the structure factor amplitudes derived from electron cryomicroscopic images of icosahedral virus particles. Using 400-kV spot-scan images of the bacteriophage P22 procapsid, we have calculated an amplitude contrast of 8.0 +/- 5.2%. The amplitude decay parameter has been estimated to be 523 +/- 188 A2 with image noise compensation and 44 +/- 66 A2 without it. These results can also be used to estimate the minimum number of virus particles needed for reconstruction at different resolutions.

Bacteriophage P22↗

PubMed1999

Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids.

Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid.

Bacteriophage P22↗

PubMed1998

Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.

Assembly of bacteriophage P22 procapsids requires the participation of approximately 300 molecules of scaffolding protein in addition to the 420 coat protein subunits. In the absence of the scaffolding, the P22 coat protein can assemble both wild-type-size and smaller size closed capsids. Both sizes of procapsid assembled in the absence of the scaffolding protein have been studied by electron cryomicroscopy. These structural studies show that the larger capsids have T = 7 icosahedral lattices and appear the same as wild-type procapsids. The smaller capsids possess T = 4 icosahedral symmetry. The two procapsids consist of very similar penton and hexon clusters, except for an increased curvature present in the T = 4 hexon. In particular, the pronounced skewing of the hexons is conserved in both sizes of capsid. The T = 7 procapsid has a local non-icosahedral twofold axis in the center of the hexon and thus contains four unique quasi-equivalent coat protein conformations that are the same as those in the T = 4 procapsid. Models of how the scaffolding protein may direct these four coat subunit types into a T = 7 rather than a T = 4 procapsid are presented.

Bacteriophage P22↗

PubMed1997

Improved common line-based icosahedral particle image orientation estimation algorithms.

Modifications are described for the center and angular parameter estimation algorithms of common line-based particle image orientation determination which is an essential step in the three-dimensional reconstruction of icosahedral virus particles. The modifications incorporate a variety of image processing, pattern recognition, and statistical tools resulting in objective and automated orientation estimation algorithms. The modified algorithms were tested using electron cryo-microscopic particle images of three different virus specimens, with sizes 400-1250 A in diameter, covering a broad range of defocus values. Evaluation of these modified algorithms shows significant improvement over the previous algorithms. The center and angular parameters were estimated with higher accuracy allowing the identification of a larger number of particle orientations. Usage of the modified estimation algorithms resulted in the identification of particle orientations which could not to be identified using the algorithms before modification. Furthermore, these improvements have resulted in the determination of a better quality and a higher resolution three-dimensional reconstruction. The improved algorithms have been developed into a software package which can be obtained via the world wide web at http://ncmi.bioch.bcm.tmc.edu/pthuman.

Algorithms↗

PubMed1996

Three-dimensional structure of scaffolding-containing phage p22 procapsids by electron cryo-microscopy.

The procapsids of bacterial viruses are the products of the polymerization of coat and scaffolding subunits, as well as the precursors in DNA packaging. Electron cryo-microscopy has been used to study the three-dimensional structures of bacteriophage P22 procapsids containing wild-type and mutant scaffolding proteins. The scaffolding mutant structure has been resolved to 19 A resolution and agrees with the 22 A resolution wild-type procapsid reconstruction. Both procapsid reconstructions contain an outer icosahedral coat protein shell and an inner scaffolding protein core. The outer core protein forms a T = 7 icosahedral lattice with distinctive channels present at the centers of the pentons and hexons. In addition, the hexons display a prominent skew. Computational isolation of the skewed hexon shows the presence of a local 2-fold axis that reduces the number of unique conformations in the asymmetric unit to four at this resolution. We have classified the four unique subunits into three distinct classes, based upon the shape of the upper domain and the presence of a channel leading to the inner coat protein surface. In addition, at the inner surface of the coat protein, finger-like regions that extend towards the scaffolding protein core are present in two of the subunits. The finger-like regions suggest the presence of an ordered interaction between the inner coat protein and the scaffolding protein. However, an icosahedral scaffolding protein shell is not formed, and the innermost scaffolding protein core does not pack with icosahedral symmetry.

Bacteriophage P22↗

PubMedDate not supplied

PTOOL: a software package for the selection of particles from electron cryomicroscopy spot-scan images.

PTOOL, a suite of tools packaged in an X Window System-based graphical user interface has been developed to allow consistent and accurate selection of single particles from low-contrast spot-scan and flood beam electron microscopy images. These user friendly and computationally efficient tools run on Unix-based workstations. Particles are selected using an automatic reference based selection method which incorporates extensive image preprocessing. An image division and subsequent image sewing mechanism allows for efficient particle selection independent of micrograph size. A particle viewing tool is provided which displays galleries of images and has options for performing image processing operations such as Fourier filtering, histogram equalization, and image masking. In addition, a tool is included to perform semiautomatic focal pair particle matching to identify the same particles on two micrographs.

Bacteriophage P22↗