We present the first three-dimensional reconstruction of a prolate, tailed phage, and its empty prohead precursor by cryo-electron microscopy. The head–tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ within the Bacillus subtilis dsDNA phage φ29. The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA. The prolate head of φ29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T=3 quasi-symmetry. Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.
