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1.
J Struct Biol ; 156(2): 334-41, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16857386

ABSTRACT

Cryoelectron tomography (CET) combines the potential of three-dimensional (3D) imaging with a close-to-life preservation of biological samples. It allows the examination of large and stochastically variable structures, such as organelles or whole cells. At the current resolution it becomes possible to visualize large macromolecular complexes in their functional cellular environments. Pattern recognition methods can be used for a systematic interpretation of the tomograms; target molecules are identified and located based on their structural signature and their correspondence with a template. Here, we demonstrate that such an approach can be used to map 70S ribosomes in an intact prokaryotic cell (Spiroplasma melliferum) with high fidelity, in spite of the low signal-to-noise ratio (SNR) of the tomograms. At a resolution of 4.7 nm the average generated from the 236 ribosomes found in a tomogram is in good agreement with high resolution structures of isolated ribosomes as obtained by X-ray crystallography or cryoelectron microscopy. Under the conditions of the experiment (logarithmic growth phase) the ribosomes are evenly distributed throughout the cytosol, occupying approximately 5% of the cellular volume. A subset of about 15% is found in close proximity to and with a distinct orientation with respect to the plasma membrane. This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes.


Subject(s)
Cryoelectron Microscopy/methods , Imaging, Three-Dimensional/methods , Ribosomes/diagnostic imaging , Tomography, X-Ray Computed/methods , Algorithms , Crystallography, X-Ray , Feasibility Studies , Macromolecular Substances/analysis , Models, Molecular , Spiroplasma/ultrastructure
2.
J Biol Chem ; 280(36): 31378-89, 2005 Sep 09.
Article in English | MEDLINE | ID: mdl-16012171

ABSTRACT

Pseudomonas aeruginosa is an opportunistic gram-negative pathogen equipped with multiple secretion systems. The type II secretion machinery (Xcp secreton) is involved in the release of toxins and enzymes. The Xcp secreton is a multiprotein complex, and most of its components share homology with proteins involved in type IV pili biogenesis. Among them, the XcpT-X pseudopilins possess characteristics of the major constituent of the type IV pili, the pilin PilA. We have shown previously that XcpT can be assembled in a multifibrillar structure that was called the pseudopilus. By using two different microscopic approaches, we show here that the pseudopili are preferentially isolated fibers rather than tight bundles. Moreover, none of the other four pseudopilins are able to form a pseudopilus, suggesting that the assembly of such a structure is a unique property of XcpT. Moreover, we show that 5 of the 12 Xcp proteins are not required for pseudopilus biogenesis, whereas they are for type II secretion. Most interestingly, we showed that one pseudopilin, XcpX, controls the assembly of XcpT into a pseudopilus. Indeed, when the number of XcpX subunits increases, the length of the pseudopilus decreases. Conversely, in the absence of XcpX, the pseudopilus length is abnormally long. Our results indicate that XcpT and XcpX directly interact with each other. Furthermore, this interaction induces a clear destabilization of XcpT. The interaction between XcpT and XcpX could be part of the molecular mechanism underlying the dynamic control of pseudopilus elongation, which could be crucial for type II-dependent protein secretion.


Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/physiology , Fimbriae, Bacterial/metabolism , Membrane Proteins/physiology , Membrane Transport Proteins/biosynthesis , Pseudomonas aeruginosa/physiology , Bacterial Adhesion/physiology , Bacterial Proteins/genetics , Cryoelectron Microscopy , Fimbriae, Bacterial/ultrastructure , Membrane Proteins/genetics , Membrane Transport Proteins/genetics , Microscopy, Electron, Transmission , Pseudomonas aeruginosa/ultrastructure
3.
Science ; 307(5708): 436-8, 2005 Jan 21.
Article in English | MEDLINE | ID: mdl-15662018

ABSTRACT

Evidence has accumulated recently that not only eukaryotes but also bacteria can have a cytoskeleton. We used cryo-electron tomography to study the three-dimensional structure of Spiroplasma melliferum cells in a close-to-native state at approximately 4-nanometer resolution. We showed that these cells possess two types of filaments arranged in three parallel ribbons underneath the cell membrane. These two filamentous structures are built of the fibril protein and possibly the actin-like protein MreB. On the basis of our structural data, we could model the motility modes of these cells and explain how helical Mollicutes can propel themselves by means of coordinated length changes of their cytoskeletal ribbons.


Subject(s)
Cytoskeleton/ultrastructure , Spiroplasma/ultrastructure , Bacterial Proteins/analysis , Blotting, Western , Cell Membrane/ultrastructure , Computer Simulation , Cryoelectron Microscopy , Cytoskeleton/chemistry , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Models, Biological , Movement , Spiroplasma/chemistry , Spiroplasma/physiology , Tomography
4.
Exp Cell Res ; 301(1): 38-42, 2004 Nov 15.
Article in English | MEDLINE | ID: mdl-15501443

ABSTRACT

Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3-D) imaging at molecular resolution (<5 nm) with a close-to-life preservation of the specimen. In conjunction with pattern recognition techniques, it enables us to map the molecular landscape inside cells. The application of cryo-ET to intact cells provides novel insights into the structure and the spatial organization of the cytoskeleton in prokaryotic and eukaryotic cells.


Subject(s)
Cryoelectron Microscopy/methods , Cytoskeleton/chemistry , Eukaryotic Cells/ultrastructure , Imaging, Three-Dimensional , Prokaryotic Cells/ultrastructure , Actin Cytoskeleton/chemistry , Animals , Cryoelectron Microscopy/instrumentation , Eukaryotic Cells/chemistry , Humans , Prokaryotic Cells/chemistry , Tomography
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