Acta Cryst. (2000). D56, 1187±1190 Badasso et al.  Head±tail connector of bacteriophage '29 1187 crystallization papers Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Purification, crystallization and initial X-ray analysis of the head±tail connector of bacteriophage u29 Mohammed O. Badasso, a Petr G. Leiman, b Yizhi Tao, b ² Yongning He, b Douglas H. Ohlendorf, c Michael G. Rossmann b * and Dwight Anderson a a Department of Microbiology and Oral Science, University of Minnesota, Minneapolis, MN 55455, USA, b Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA, and c Department of Biochemistry, University of Minnesota, Minneapolis, MN 55455, USA ² Current address: Department of Molecular and Cell Biology, Harvard University, Cambridge, MA 02138, USA. Correspondence e-mail: mgr@indiana.bio.purdue.edu # 2000 International Union of Crystallography Printed in Denmark ± all rights reserved The head±tail connector of bacteriophage '29, an oligomer of gene product 10 (gp10), was crystallized into various forms. The most useful of these were an orthorhombic P22 1 2 1 form (unit-cell parameters a = 143.0, b = 157.0, c = 245.2 A Ê ), a monoclinic C2 form (a = 160.7, b = 143.6, c = 221.0 A Ê ,  = 97.8  ) and another monoclinic C2 form (a = 177.0, b = 169.1, c = 185.2 A Ê ,  = 114.1  ). Frozen crystals diffracted to about 3.2 A Ê resolution. There is one connector per crystallographic asymmetric unit in each case. Rotation functions show the connector to be a dodecamer. Translation functions readily determined the position of the 12-fold axis in each unit cell. The structure is being determined by 12-fold electron-density averaging within each crystal and by averaging between the various crystal forms. Received 4 April 2000 Accepted 26 June 2000 1. Introduction The Bacillus subtilis bacteriophage '29 is a small double-stranded DNA (19 kbp) virus with a prolate head and complex structure (Anderson et al., 1966). Because DNA pack- aging in vitro rivals in vivo assembly in ef®ciency, '29 is a leading system for study of the DNA packaging mechanism. The '29 prohead, a precursor capsid into which DNA is packaged, consists of only four structural proteins. These are the head±tail connector (gene product 10; gp10), scaffolding protein (gp7), major capsid protein (gp8) and head ®bers (gp8.5) (Me  ndez et al., 1971). A cyclic hexamer of a 174-base '29-encoded RNA, prohead RNA (pRNA), is bound to the connector and is needed for DNA packaging (Guo et al. , 1998; Hendrix, 1998; Zhang et al., 1998). The connector, pRNA and the ATPase gp16 comprise the DNA-packaging machine; analysis of the structure of these components is needed in order to determine the packaging mechanism. The '29 head±tail connector is a cone- shaped oligomer of the 35.9 kDa gp10 (Carazo et al., 1986; Tsuprun et al., 1994; Mu È ller et al., 1997; Valpuesta et al., 1999). The connector occupies the vertex at the base of both the prohead and mature head, which are extended icosahedrons having an elongation number of Q = 5 and 235 subunits (Tao et al. , 1998). The connector has a diameter ranging from 140 to 80 A Ê , with an axial channel of 60±35 A Ê that is large enough to accommodate DNA passage (Tao et al. , 1998). Free connectors wrap supercoiled DNA in a single turn around their circumference, restraining a negative supercoil, and wrapping of DNA by the connector of the prohead may provide a stable initiation complex for packaging (Turnquist et al., 1992). In addition to its DNA-translocation function, the connector functions in the initiation of prohead assembly (Guo et al. , 1991; Anderson & Reilly, 1993). The three-dimensional X-ray structure analysis of the connector protein is important in understanding these functions. Connectors of double-stranded DNA phages, such as T3 (Carazo et al. , 1996), T4 (Driedonks et al. , 1981), T7 (Kocsis et al. , 1985),  (Kochan et al., 1984), P22 (Bazinet & King, 1985) and '29 (Carrascosa et al., 1982; Guasch et al., 1998; Valle et al. , 1999), have all shown 12-fold symmetry, although 13-fold symmetry has also been reported (Dube et al. , 1993; Tsuprun et al., 1994). These investigations have been based on negative staining, cryo-electron microscopy and image processing, as well as atomic force microscopy. It is clear that '29 connector interactions with proteins gp8 of the head and gp11 of the neck, pRNA, the DNA- translocating ATPase gp16 and DNA would be better understood if high-resolution X-ray data were available. In this paper, we describe the crystallization conditions that yielded well ordered crystals of '29 connectors suitable for X-ray analysis and the preliminary crystal- lographic characterization of these crystals. 2. Materials and methods 2.1. Protein expression and purification The connectors of bacteriophage '29 were assembled in Escherichia coli (pPC28D1) that expresses gp10 (GarcõÂa et al., 1984; Ibanez et