Three-dimensional structure of T3 connector purified from overexpressing bacteria.
The bacteriophage T3 connector has been purified from overexpressed protein in Escherichia coli, harboring a plasmid containing the gene encoding p8 protein. The connector, which is composed of 12 copies of p8, has been crystallized in two-dimensional sheets and studied by electron microscopy from negatively stained specimens. A two-dimensional Fourier filtering and averaging procedure was performed with crystalline specimens. In addition, single particle averaging techniques were used with other preparations. The average images obtained from these two approaches gave similar results. A three-dimensional reconstruction from two-dimensional crystals of T3 connectors was obtained by collecting several sets of tilted views and using standard Fourier procedures. The resolution of the three-dimensional map was 1.65 nm. The reconstructed connector shows two main domains: a wider one with 12 small units in the periphery and with an external diameter of 14.9 nm, and a smaller one with 8.5 nm diameter. The height of the reconstructed connector has been determined to be around 8.5 nm. The reconstruction clearly shows an internal open channel running along the longitudinal axis of the particle and having an average diameter of 3.7 nm.
Valpuesta
J M
JM
Centro Nacional de Biotecnología, C.S.I.C. Universidad Autónoma de Madrid, Spain.
Fujisawa
H
H
Marco
S
S
Carazo
J M
JM
Carrascosa
J L
JL
D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005583:Fourier Analysis; D015964:Gene Expression Regulation, Bacterial; D008854:Microscopy, Electron; D010957:Plasmids; D015183:Restriction Mapping; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure
D036022:Capsid Proteins; D014764:Viral Proteins; C073670:p8 protein, Bacteriophage T3