Preliminary crystallographic analysis of the bacteriophage P22 portal protein.
Portal proteins are components of large oligomeric dsDNA pumps connecting the icosahedral capsid of tailed bacteriophages to the tail. Prior to the tail attachment, dsDNA is actively pumped through a central cavity formed by the subunits. We have studied the portal protein of bacteriophage P22, which is the largest connector characterized among the tailed bacteriophages. The molecular weight of the monomer is 82.7 kDa, and it spontaneously assembles into an oligomeric structure of approximately 1.0 MDa. Here we present a preliminary biochemical and crystallographic characterization of this large macromolecular complex. The main difficulties related to the crystallization of P22 portal protein lay in the intrinsic dynamic nature of the portal oligomer. Recombinant connectors assembled from portal monomers expressed in Escherichia coli form rings of different stoichiometry in solution, which cannot be separated on the basis of their size. To overcome this intrinsic heterogeneity we devised a biochemical purification that separates different ring populations on the basis of their charge. Small ordered crystals were grown from drops containing a high concentration of the kosmotropic agent tert-butanol and used for data collection. A preliminary crystallographic analysis to 7.0-A resolution revealed that the P22 portal protein crystallized in space group I4 with unit cell dimensions a=b=409.4A, c=260.4A. This unit cell contains a total of eight connectors. Analysis of the noncrystallographic symmetry by the self-rotation function unambiguously confirmed that bacteriophage P22 portal protein is a dodecamer with a periodicity of 30 degrees. The cryo-EM reconstruction of the dodecahedral bacteriophage T3 portal protein will be used as a model to initiate phase extension and structure determination.
Cingolani
Gino
G
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Moore
Sean D
SD
Prevelige
Peter E
PE
Johnson
John E
JE
D017100:Bacteriophage P22 / Q000737:chemistry*; D001435:Bacteriophages / Q000378:metabolism*; D002412:Cations; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D017356:Synchrotrons; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology; D019065:Virus Assembly; D014965:X-Rays; D020002:tert-Butyl Alcohol / Q000494:pharmacology
D002412:Cations; D014764:Viral Proteins; D004247:DNA; D020002:tert-Butyl Alcohol