Crystallization and preliminary X-ray characterization of the relaxase domain of F factor TraI.
Conjugative plasmids are capable of transferring a copy of themselves in single-stranded form from donor to recipient bacteria. Prior to transfer, one plasmid strand must be cleaved in a sequence-specific manner by a relaxase or mobilization protein. TraI is the relaxase for the conjugative plasmid F factor. A 36 kDa N-terminal fragment of TraI possesses the single-stranded DNA-binding and cleavage activity of the protein. Crystals of the 36 kDa TraI fragment in native and selenomethionine-labeled forms were grown by sitting-drop vapor-diffusion methods using PEG 1000 as the precipitant. Crystallization in the presence of chloride salts of magnesium and strontium was required to obtain crystals yielding high-resolution diffraction. To maintain high-resolution diffraction upon freezing, crystals had to be soaked in crystallization buffer with stepwise increases of ethylene glycol. The resulting crystals were trigonal and diffracted to a resolution of 3.1 A or better using synchrotron radiation.
Larkin
Chris
C
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Datta
Saumen
S
Nezami
Azin
A
Dohm
Julie A
JA
Schildbach
Joel F
JF
D001665:Binding Sites; D003460:Crystallization; D004265:DNA Helicases / Q000737:chemistry*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004058:Diffusion; D029968:Escherichia coli Proteins; D019855:Ethylene Glycol / Q000737:chemistry; D005144:F Factor / Q000737:chemistry*; D005615:Freezing; D008274:Magnesium / Q000737:chemistry; D015636:Magnesium Chloride / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012645:Selenomethionine / Q000737:chemistry; D014961:X-Ray Diffraction; D014965:X-Rays
D029968:Escherichia coli Proteins; D015636:Magnesium Chloride; D011092:Polyethylene Glycols; D012645:Selenomethionine; D004254:DNA Nucleotidyltransferases; C110340:DNA relaxase; C027093:TraI protein, E coli; D004265:DNA Helicases; D019855:Ethylene Glycol; D008274:Magnesium