Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition.

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.

[1]  A S Frangakis,et al.  Noise reduction in electron tomographic reconstructions using nonlinear anisotropic diffusion. , 2001, Journal of structural biology.

[2]  V. Lučić,et al.  Structural studies by electron tomography: from cells to molecules. , 2005, Annual review of biochemistry.

[3]  F. Neidhardt,et al.  Escherichia Coli and Salmonella: Typhimurium Cellular and Molecular Biology , 1987 .

[4]  F. Schluenzen,et al.  Structure of Functionally Activated Small Ribosomal Subunit at 3.3 Å Resolution , 2000, Cell.

[5]  T. Steitz,et al.  Crystal Structure of the Large Ribosomal Subunit at 5-Angstrom Resolution , 2000 .

[6]  F. Förster,et al.  Identification of macromolecular complexes in cryoelectron tomograms of phantom cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Florence Tama,et al.  Structure of the E. coli protein-conducting channel bound to a translating ribosome , 2005, Nature.

[8]  P. Graumann,et al.  Specific polar localization of ribosomes in Bacillus subtilis depends on active transcription , 2001, EMBO reports.

[9]  A. Roseman.  Particle finding in electron micrographs using a fast local correlation algorithm. , 2003, Ultramicroscopy.

[10]  Wolfgang Baumeister,et al.  A visual approach to proteomics , 2006, Nature Reviews Molecular Cell Biology.

[11]  F. Förster,et al.  Nuclear Pore Complex Structure and Dynamics Revealed by Cryoelectron Tomography , 2004, Science.

[12]  T. Earnest,et al.  Crystal Structure of the Ribosome at 5.5 Å Resolution , 2001, Science.

[13]  J. Frank,et al.  Solution Structure of the E. coli 70S Ribosome at 11.5 Å Resolution , 2000, Cell.

[14]  T Aschenbrenner,et al.  Scaling-index method as an image processing tool in scanning-probe microscopy. , 2001, Ultramicroscopy.

[15]  J. Holton,et al.  Structures of the Bacterial Ribosome at 3.5 Å Resolution , 2005, Science.

[16]  J. Dubochet,et al.  Cryo-electron microscopy of vitrified specimens , 1988, Quarterly Reviews of Biophysics.

[17]  Hegerl,et al.  The EM Program Package: A Platform for Image Processing in Biological Electron Microscopy , 1996, Journal of structural biology.

[18]  J. Frank,et al.  Three-dimensional reconstruction with contrast transfer function correction from energy-filtered cryoelectron micrographs: procedure and application to the 70S Escherichia coli ribosome. , 1997, Journal of structural biology.

[19]  Wolfgang Baumeister,et al.  Three-Dimensional Structure of Herpes Simplex Virus from Cryo-Electron Tomography , 2003, Science.

[20]  T. Steitz,et al.  The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. , 2000, Science.

[21]  Frank Schluenzen,et al.  High Resolution Structure of the Large Ribosomal Subunit from a Mesophilic Eubacterium , 2001, Cell.

[22]  J. Errington,et al.  Compartmentalization of transcription and translation in Bacillus subtilis , 2000, The EMBO journal.

[23]  C. Condon,et al.  Construction and Initial Characterization of Escherichia coli Strains with Few or No Intact Chromosomal rRNA Operons , 1999, Journal of bacteriology.

[24]  D. Fletcher,et al.  Spiroplasma Swim by a Processive Change in Body Helicity , 2005, Cell.

[25]  Friedrich Förster,et al.  TOM software toolbox: acquisition and analysis for electron tomography. , 2005, Journal of structural biology.

[26]  B. Alberts.  The Cell as a Collection of Protein Machines: Preparing the Next Generation of Molecular Biologists , 1998, Cell.

[27]  J. Forchhammer,et al.  Growth rate of polypeptide chains as a function of the cell growth rate in a mutant of Escherichia coli 15. , 1971, Journal of molecular biology.

[28]  H. Bremer.  Modulation of Chemical Composition and Other Parameters of the Cell by Growth Rate , 1999 .

[29]  Achilleas S. Frangakis,et al.  Cryo-Electron Tomography Reveals the Cytoskeletal Structure of Spiroplasma melliferum , 2005, Science.

[30]  C. Vonrhein,et al.  Structure of the 30S ribosomal subunit , 2000, Nature.

[31]  W. Baumeister,et al.  Macromolecular Architecture in Eukaryotic Cells Visualized by Cryoelectron Tomography , 2002, Science.

[32]  F. Förster,et al.  Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[33]  A S Frangakis,et al.  Toward detecting and identifying macromolecules in a cellular context: template matching applied to electron tomograms. , 2000, Proceedings of the National Academy of Sciences of the United States of America.