Motors generating mechanical force, powered by the hydrolysis of ATP, translocate doublestranded DNA into preformed capsids (proheads) of bacterial viruses 1,2 and certain animal viruses 3 . Here we describe the motor that packages the double-stranded DNA of the Bacillus subtilis bacteriophage ϕ29 into a precursor capsid. We determined the structure of the head-tail connector-the central component of the ϕ29 DNA packaging motor-to 3.2Å resolution by means of X-ray crystallography. We then fitted the connector into the electron densities of the prohead and of the partially packaged prohead as determined using cryo-electron microscopy and image reconstruction analysis. Our results suggest that the prohead plus dodecameric connector, prohead RNA, viral ATPase and DNA comprise a rotary motor with the head-prohead RNAATPase complex acting as a stator, the DNA acting as a spindle, and the connector as a ball-race. The helical nature of the DNA converts the rotary action of the connector into translation of the DNA.The bacteriophage ϕ29 (Fig. 1) is a 19-kilobase (19-kb) double-stranded DNA (dsDNA) virus with a prolate head and complex structure 4 . The prohead (Fig. 1), into which the DNA is packaged, is about 540Å long and 450Å wide 5 . The ϕ29 connector, a cone-shaped dodecamer of gene product 10 (gp10), occupies the pentagonal vertex at the base of the prohead 5 and is the portal for DNA entry during packaging and DNA ejection during infection 6 . The connector, in association with the oligomeric, ϕ29-encoded prohead RNA (pRNA) and a viral ATPase (gp16), is required for DNA packaging [7][8][9] . However, only the first 120 bases of the 174-base pRNA are essential for packaging 7 the genomic dsDNA with gp3 (DNA-gp3) can be packaged into proheads in about three minutes in vitro (P.J.J., unpublished results). The connector proteins of tailed phages 6 vary in relative molecular mass (M r ) from 36,000 (36K) in ϕ29 to 83K in phage P22, and assemble into oligomers with a central channel. The structure of the isolated ϕ29 connector has been studied by atomic force microscopy 10 and cryo-electron microscopy (cryo-EM) of two-dimensional arrays 11 , immuno-electron microscopy 12 and X-ray crystallography 13,14 .The connector structure, as now determined by X-ray crystallography, can be divided into three, approximately cylindrical regions: the narrow end, the central part, and the wide end, having external radii (Å ) of 33, 47 and 69, respectively (Fig. 2). These regions are respectively 25, 28 and 22Å in height, making the total connector 75Å long. The internal channel has a diameter of about 36Å at the narrow end, increasing to 60Å at the wide end.Comparison with electron microscopy reconstructions 5,11 shows that the narrow end protrudes from the portal vertex of the phage head, is associated with the multimeric pRNA, and binds the lower collar in the mature virus.The electron density of the connector was interpreted in terms of the amino-acid sequence 15 and was confirmed by the two Hg sites (see Methods section)...

Three-dimensional structures of the double-stranded DNA bacteriophage phi29 scaffolding protein (gp7) before and after prohead assembly have been determined at resolutions of 2.2 and 2.8 A, respectively. Both structures are dimers that resemble arrows, with a four-helix bundle composing the arrowhead and a coiled coil forming the tail. The structural resemblance of gp7 to the yeast transcription factor GCN4 suggests a DNA-binding function that was confirmed by native gel electrophoresis. DNA binding to gp7 may have a role in mediating the structural transition from prohead to mature virus and scaffold release. A cryo-EM analysis indicates that gp7 is arranged inside the capsid as a series of concentric shells. The position of the higher density features in these shells correlates with the positions of hexamers in the equatorial region of the capsid, suggesting that gp7 may regulate formation of the prolate head through interactions with these hexamers.

The head±tail connector of bacteriophage 929 is composed of 12 36 kDa subunits with 12-fold symmetry. It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads. This motor consists of the head±tail connector, surrounded by a 929-encoded, 174-base, RNA and a viral ATPase protein, both of which have ®vefold symmetry in three-dimensional cryo-electron microscopy reconstructions. DNA is translocated into the prohead through a 36 A Ê diameter pore in the center of the connector, where the DNA takes the role of a motor spindle. The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA. The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A Ê resolution model derived from cryo-electron microscopy. The model phases were extended to 3.5 A Ê resolution using 12-fold non-crystallographic symmetry averaging and solvent¯attening. Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative. The resultant 3.2 A Ê single isomorphous replacement phases were improved using density modi®cation, producing an interpretable electron-density map. The crystallographically re®ned structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule. One of these was in the same space group and almost isomorphous, whereas the other was in space group P2 1 2 1 2. The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively¯exible, particularly in the protruding domain at the wide end of the connector. This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging.

X-ray analyses have defined the three-dimensional structures of crystals of mouse and human renins complexed with peptide inhibitors at resolutions of 1.9 and 2.8 A, respectively. The exquisite specificity of renin arises partly from ordered loop regions at the periphery of the binding cleft. Although the pattern of main-chain hydrogen bonding in other aspartic proteinase inhibitor complexes is conserved in renins, differences in the positions of secondary structure elements (particularly helices) also lead to improved specificity in renins for angiotensinogen substrates.

No abstract