The three-dimensional structure of a DNA translocating machine at 10 A resolution.

BACKGROUND Head-tail connectors are viral substructures that are very important in the viral morphogenetic cycle, having roles in the formation of the precursor capsid (prohead), DNA packaging, tail binding to the mature head and in the infection process. Structural information on the connector would, therefore, help us to understand how this structure is related to a multiplicity of functions. RESULTS Recombinant bacteriophage phi29 connectors have been crystallized in two-dimensional aggregates. An average projection image and a three-dimensional map have been obtained at 8 A and 10 A resolution, respectively, from untilted and tilted images of vitrified specimens of the two-dimensional crystals. The average projection image reveals a central mass surrounding a channel with 12 appendages protruding from the central mass. The three-dimensional map reveals a wide domain surrounded by 12 appendages that interact with the prohead vertex, and a narrow domain that interacts with the bacteriophage tail. At the junction of the two domains, 12 smaller appendages are visualized. A channel runs along the axis of the connector structure and is sufficiently wide to allow a double-stranded DNA molecule to pass through. CONCLUSIONS The propeller-like structure of the phi29 connector strengthens the notion of the connector rotating during DNA packaging. The groove formed by the two lanes of large and small appendages may act as a rail to prevent the liberation of the connector from the prohead vertex during rotation.

[1]  J. García,et al.  Cloning, nucleotide sequence and high level expression of the gene coding for the connector protein of Bacillus subtilis phage φ 29 , 1984 .

[2]  R Dargahi,et al.  SPECTRA: a system for processing electron images of crystals. , 1993, Ultramicroscopy.

[3]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[4]  J. Carazo,et al.  Three-dimensional structure of T3 connector purified from overexpressing bacteria. , 1992, Journal of molecular biology.

[5]  L. E. Donate,et al.  Study of two related configurations of the neck of bacteriophage Ø29 , 1990 .

[6]  J. García,et al.  Overproduction and purification of the connector protein of Bacillus subtilis phage phi 29. , 1984, Nucleic acids research.

[7]  M F Schmid,et al.  A brief description of I.C.E.: the integrated crystallographic environment. , 1996, Journal of structural biology.

[8]  José-Jesús Fernández,et al.  A spectral estimation approach to contrast transfer function detection in electron microscopy , 1997 .

[9]  H Murialdo,et al.  Bacteriophage lambda DNA: the beginning of the end , 1990, Journal of bacteriology.

[10]  G. Schertler,et al.  Low resolution structure of bovine rhodopsin determined by electron cryo-microscopy. , 1995, Biophysical journal.

[11]  S. Grimes,et al.  The bacteriophage phi29 packaging proteins supercoil the DNA ends. , 1997, Journal of molecular biology.

[12]  C. Zhang,et al.  Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation. , 1998, Molecular cell.

[13]  J. Lepault,et al.  Structure of purple membrane from halobacterium halobium: recording, measurement and evaluation of electron micrographs at 3.5 Å resolution , 1986 .

[14]  Marco,et al.  Xmipp: An Image Processing Package for Electron Microscopy , 1996, Journal of structural biology.

[15]  R Henderson,et al.  Analysis of electron microscope images and electron diffraction patterns of thin crystals of phi 29 connectors in ice. , 1994, Journal of molecular biology.

[16]  Peixuan Guo,et al.  Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29. , 1987, Journal of molecular biology.

[17]  Roger W. Hendrix,et al.  Control Mechanisms in dsDNA Bacteriophage Assembly , 1988 .

[18]  R. Henderson,et al.  Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. , 1990, Journal of molecular biology.

[19]  F. Major,et al.  Function of hexameric RNA in packaging of bacteriophage phi 29 DNA in vitro. , 1998, Molecular cell.

[20]  Timothy S Baker,et al.  Assembly of a Tailed Bacterial Virus and Its Genome Release Studied in Three Dimensions , 1998, Cell.

[21]  J M Carazo,et al.  Three-dimensional reconstruction of the connector of bacteriophage phi 29 at 1.8 nm resolution. , 1986, Journal of molecular biology.

[22]  R A Crowther,et al.  Harmonic analysis of electron microscope images with rotational symmetry. , 1971, Journal of molecular biology.

[23]  L. E. Donate,et al.  Bacteriophage T3 connector: three-dimensional structure and comparison with other viral head-tail connecting regions. , 1988, Journal of molecular biology.

[24]  J. Pous,et al.  Crystallographic analysis reveals the 12-fold symmetry of the bacteriophage phi29 connector particle. , 1998, Journal of molecular biology.

[25]  R A Crowther,et al.  MRC image processing programs. , 1996, Journal of structural biology.

[26]  L. E. Donate,et al.  Role of the Amino-terminal Domain of Bacteriophage # 29 Connector in DNA Binding and Packaging , 2001 .

[27]  P. Guo,et al.  A small viral RNA is required for in vitro packaging of bacteriophage phi 29 DNA. , 1987, Science.

[28]  M van Heel,et al.  A new generation of the IMAGIC image processing system. , 1996, Journal of structural biology.

[29]  J. Caldentey,et al.  Gene 20 product of bacteriophage T4. II. Its structural organization in prehead and bacteriophage. , 1983, Journal of molecular biology.

[30]  D. Anderson,et al.  Analysis of gene function of bacteriophage phi 29 of Bacillus subtilis: identification of cistrons essential for viral assembly , 1976, Journal of virology.

[31]  P. Bullough,et al.  High-resolution spot-scan electron microscopy of microcrystals of an alpha-helical coiled-coil protein. , 1990, Journal of molecular biology.

[32]  R. Hendrix,et al.  Symmetry mismatch and DNA packaging in large bacteriophages. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[33]  L. Black.  DNA packaging in dsDNA bacteriophages. , 1989, Annual review of microbiology.

[34]  J M Carazo,et al.  Analysis of structural variability within two-dimensional biological crystals by a combination of patch averaging techniques and self organizing maps. , 1996, Ultramicroscopy.

[35]  J. Carazo,et al.  Three-dimensional reconstruction of bacteriophage φ29 neck particles at 2.2 nm resolution☆ , 1985 .

[36]  J. Carazo,et al.  Structural localizaton of the proteins of the head to tail connecting region of bacteriophage φ29 , 1983 .

[37]  B Sheehan,et al.  AVS software for visualization in molecular microscopy. , 1996, Journal of structural biology.

[38]  R. Markham,et al.  Methods for the enhancement of image detail and accentuation of structure in electron microscopy , 1963 .

[39]  R. Mellado,et al.  Assembly of Bacillus subtilis phage phi29. 1. Mutants in the cistrons coding for the structural proteins. , 1977, European journal of biochemistry.

[40]  D A Agard,et al.  A least-squares method for determining structure factors in three-dimensional tilted-view reconstructions. , 1983, Journal of molecular biology.

[41]  A. Engel,et al.  The bacteriophage φ29 head–tail connector imaged at high resolution with the atomic force microscope in buffer solution , 1997, The EMBO journal.

[42]  J. Carrascosa,et al.  Structure of viral connectors and their function in bacteriophage assembly and DNA packaging , 1994, Quarterly Reviews of Biophysics.

[43]  B Ermentrout,et al.  Rotary DNA motors. , 1995, Biophysical journal.

[44]  E. Egelman,et al.  The bacteriophage phi 29 head-tail connector shows 13-fold symmetry in both hexagonally packed arrays and as single particles. , 1994, Biophysical journal.

[45]  P. Guo,et al.  Initiation events in in-vitro packaging of bacteriophage phi 29 DNA-gp3. , 1987, Journal of molecular biology.

[46]  M. Heel,et al.  The portal protein of bacteriophage SPP1: a DNA pump with 13‐fold symmetry. , 1993, The EMBO journal.

[47]  Sherwood R. Casjens,et al.  DNA packaging by the double-stranded DNA bacteriophages , 1980, Cell.

[48]  P. Shaw,et al.  Tilted specimen in the electron microscope: A simple specimen holder and the calculation of tilt angles for crystalline specimens , 1981 .

[49]  R. Henderson,et al.  Three-dimensional structure determination by electron microscopy of two-dimensional crystals. , 1982, Progress in biophysics and molecular biology.

[50]  R. Hendrix.  Bacteriophage DNA Packaging RNA Gears in a DNA Transport Machine , 1998, Cell.