MRC Image Processing Programs

Crowther, R.A.; Henderson, Richard A.; Smith, Judith M.

doi:10.1006/jsbi.1996.0003

Cited by 704 publications

(535 citation statements)

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“…Electron micrographs used for image processing were photographed at magnifications between 25 000 and 63 000. They were scanned with a Zeiss SCAI scanner at 28 or 14 micrometer step size and processed using the MRC package of programs (Crowther et al, 1996). After spline-fitting a line down the axis of each image of a curved polymer, unbending was achieved by bilinear interpolation (Egelman, 1986).…”

Section: Methodsmentioning

confidence: 99%

Helical Tubes of FtsZ from Methanococcus jannaschii

Löwe¹,

Amos²

2000

Biological Chemistry

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Bacterial cell division depends on the formation of a cytokinetic ring structure, the Z-ring. The bacterial tubulin homologue FtsZ is required for Z-ring formation. FtsZ assembles into various polymeric forms in vitro, indicating a structural role in the septum of bacteria. We have used recombinant FtsZ1 protein from M. jannaschii to produce helical tubes and sheets with high yield using the GTP analogue GMPCPP [guanylyl-(␣,␤)-methylene-diphosphate]. The sheets appear identical to the previously reported Ca ++ -induced sheets of FtsZ from M. jannaschii that were shown to consist of 'thick'-filaments in which two protofilaments run in parallel. Tubes assembled either in Ca ++ or in GMPCPP contain filaments whose dimensions indicate that they could be equivalent to the 'thick'-filaments in sheets. Some tubes are hollow but others are filled by additional protein density. Helical FtsZ tubes differ from eukaryotic microtubules in that the filaments curve around the filament axis with a pitch of ~ 430 Å for Ca ++ -induced tubes or 590 -620 Å for GMPCPP. However, their assembly in vitro as wellordered polymers over distances comparable to the inner circumference of a bacterium may indicate a role in vivo. Their size and stability make them suitable for use in motility assays.

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Section: Methodsmentioning

confidence: 99%

Helical Tubes of FtsZ from Methanococcus jannaschii

Löwe¹,

Amos²

2000

Biological Chemistry

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“…The magnification on the CCD had previously been determined to be 2.28 Å per pixel under these conditions. Images (12,698) of the putative amidase were manually selected using Ximdisp (Crowther et al 1996). The images were aligned to the image average and subjected to principal component analysis as in Frank (2006) using the SPIDER package (Frank et al 1996).…”

Section: Fermentationmentioning

confidence: 99%

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

Makhongela

Głowacka

Agarkar

et al. 2007

Appl Microbiol Biotechnol

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An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strain Geobacillus pallidus RAPc8, was produced at high expression levels (20 U/mg) in small-scale fermentations of Escherichia coli. The enzyme was purified to 90% homogeneity with specific activity of 1,800 U/mg in just two steps, namely, heat-treatment and gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopic (EM) analysis of the homogenous enzyme showed the native enzyme to be a homohexamer of 38 kDa subunits. Analysis of the biochemical properties of the amidase showed that the optimal temperature and pH for activity were 50 and 7.0°C, respectively. The amidase exhibited high thermal stability at 50 and 60°C, with half-lives greater than 5 h at both temperatures. At 70 and 80°C, the half-life values were 43 and 10 min, respectively. The amidase catalyzed the hydrolysis of low molecular weight aliphatic amides, with D-selectivity towards lactamide. Inhibition studies showed activation/inhibition data consistent with the presence of a catalytically active thiol group. Acyl transfer reactions were demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor; the highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4°C and on Amberlite-XAD57 resulted in low protein binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking resulted in high protein binding yield and high immobilized specific activity (80% of non-immobilized activity). Characterization of Eupergit C-immobilized preparations showed that the optimum reaction temperature was unchanged, the pH range was somewhat broadened, and stability was enhanced giving half-lives of 52 min at 70°C and 30 min at 80°C. The amidase has potential for application under high temperature conditions as a biocatalyst for D-selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer.

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“…Areas were selected from micrographs by optical diffraction and digitized at 10 m raster size using a Joyce Loebl flat-bed densitometer. The digitized images, typically in areas of 1000-2500 pixels on each side, were processed using the Medical Research Council image-processing programs (Crowther et al, 1996). Briefly, square areas were boxed off and their Fourier transforms calculated, from which the lattice parameters were determined.…”

Section: Electron Microscopy and Image Processingmentioning

confidence: 99%

Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two‐dimensional crystals

Koronakis

et al. 1997

Molecular Microbiology

122

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SummaryTolC is an outer membrane protein required for the export of virulence proteins and toxic compounds without a periplasmic intermediate. We show that TolC is an integral part of the translocator, interacting with inner membrane components, by demonstrating a need for TolC in protein export not only from intact cells but also from sphaeroplasts. To establish the structure of TolC, and thus gain information on how this might be achieved, the protein was purified from the Escherichia coli outer membrane, as a trimer, and crystallized in two-dimensional lattices by reconstitution in phospholipid bilayers. The projection structure at 12 Å resolution showed a threefold symmetric molecule of 58 Å outer diameter, and a single pool of stain filling its centre. Side views parallel to the membrane plane revealed an additional domain outside the membrane. Eighteen membranespanning ␤-strands were predicted for the 51.5 kDa monomer, excluding a 7 kDa C-terminal segment, and this segment was shown to contain a proteinase K-sensitive site that was exposed in reconstituted membranes and sphaeroplasts, but which was protected in intact cells. The combined data suggest that TolC is a trimeric outer membrane protein with each monomer comprising a membrane domain, predicted to be ␤-barrel, and a C-terminal periplasmic domain. The latter could form part of the bridge to the energized inner membrane component of the translocation complex.

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MRC Image Processing Programs

Cited by 704 publications

References 0 publications

Helical Tubes of FtsZ from Methanococcus jannaschii

Helical Tubes of FtsZ from Methanococcus jannaschii

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two‐dimensional crystals

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