Three-dimensional reconstruction of imperfect two-dimensional crystals

Saxton, W. O.; Baumeister, W.; Hahn, M.

doi:10.1016/0304-3991(84)90057-3

Cited by 260 publications

(149 citation statements)

References 12 publications

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“…For cryoelectron tomography, specimens were prepared as above but cells were gently concentrated 20-fold by vacuum filtration through a 0.22-μm filter. Tilt series were acquired automatically using FEI Xplore3D software and the Saxton tilt scheme (42). The tilt range varied from ±53°to ±60°, and the total electron dose for each series was kept below 100 e − /Å 2 .…”

Section: Methodsmentioning

confidence: 99%

Micron-scale holes terminate the phage infection cycle

Dewey¹,

Savva

White³

et al. 2010

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

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Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or "holes." In the phages λ and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the λ-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 μm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.bacteriophage | cryoelectron tomography | Escherichia coli | holin | lambda B acteriophage lysis, the most frequent cytolethal event in the biosphere, is a precisely scheduled process controlled by proteins of the holin family (1). Holins are an extremely diverse class of small phage-encoded membrane proteins (2). The best studied holin is S105, a 105-residue polypeptide with three transmembrane domains (TMDs) encoded by the S gene of phage λ (3). Throughout the period of late gene expression and particle assembly, S105 accumulates in the cytoplasmic membrane of Escherichia coli without any effect on its integrity (4). Suddenly, at a programmed time, S105 triggers to form a lesion, or hole, in the membrane; this allows the λ-endolysin, R, to escape from the cytoplasm and attack the cell wall (2). In phages of Gram-negative hosts, there is a third step to complete the lysis pathway involving a protein or protein complex, the spanin, which connects the cytoplasmic and outer membranes (5, 6). In λ, the spanin complex consists of the cytoplasmic membrane protein, Rz, and the outer membrane lipoprotein, Rz1. This complex is essential for lysis in media containing millimolar concentrations of divalent cations, and thus is thought to act by disrupting the outer membrane, possibly by fusion with the inner membrane (6).Although the S105 holin has been extensively studied using genetic and biochemical approaches (3, 4, 7-9), nothing is known about the membrane holes except that they are nonspecific and large enough to allow escape of fully folded tetrameric R-β-galactosi...

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Section: Methodsmentioning

confidence: 99%

Micron-scale holes terminate the phage infection cycle

Dewey¹,

Savva

White³

et al. 2010

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

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“…Using the same subvolumes, a variance map for the final three-dimensional structure was calculated to illustrate the regions of the structure with a high degree of variance (30).…”

Section: Methodsmentioning

confidence: 99%

Three-dimensional Structure of Wza, the Protein Required for Translocation of Group 1 Capsular Polysaccharide across the Outer Membrane of Escherichia coli

Beis

Collins

Ford

et al. 2004

Journal of Biological Chemistry

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Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli. Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-Å resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein.Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits. Symmetry analysis of the three-dimensional structure combined with biochemical two-and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits. Wza is best described as a stack of two 4-Å high rings with differing diameters providing a mushroom-like aspect from the side. The larger ring has a distinctive square shape with a diameter of 115 Å, whereas the smaller is almost circular with a diameter of 90 Å. In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of ϳ40 Å in diameter. The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side. We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide.

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“…Holey grids (Quantifoil, Germany) with larger grid squares (200 mesh, 3.5/1.0 μm) were used to prepare frozen hydrated MHV-68 virion samples for tomography data collection with a specimen temperature of about -172°C. Tilt angles ranging from −60° to 60° were chosen according to the cosine rule (Saxton et al 1984), which uses decreasing angle increments for large tilting angles. The image data was recorded on CCD using the TVIPS EMMENU tomography program, with an electron dosage of ~1 electron/Å 2 /frame, and a total dosage of 60-80 electron/ Å 2 for the whole tilt series.…”

Section: Cryo-electron Tomographymentioning

confidence: 99%

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy

Dai

Jia

Bortz

et al. 2008

Journal of Structural Biology

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Gammaherpesviruses, including the human pathogens Epstein-Barr virus and Kaposi's sarcomaassociated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of the technique of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that inner layer tegument across different subfamilies of herpesviruses have evolved significantly to confer specific characteristics related to viral-host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection.

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Three-dimensional reconstruction of imperfect two-dimensional crystals

Cited by 260 publications

References 12 publications

Micron-scale holes terminate the phage infection cycle

Micron-scale holes terminate the phage infection cycle

Three-dimensional Structure of Wza, the Protein Required for Translocation of Group 1 Capsular Polysaccharide across the Outer Membrane of Escherichia coli

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy

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