Purification, crystallization and preliminary crystallographic analysis of a thermostable endonuclease IV from Thermotoga maritima.
The DNA-repair enzyme endonuclease IV from the thermophilic bacterium Thermotoga maritima MSB8 (reference sequence NC_000853) has been expressed in Escherichia coli and crystallized for X-ray analysis. T. maritima endonuclease IV is a 287-amino-acid protein with 32% sequence identity to E. coli endonuclease IV. The protein was purified to homogeneity and was crystallized using the sitting-drop vapor-diffusion method. The protein crystallized in space group P6(1), with one biological molecule in the asymmetric unit, corresponding to a Matthews coefficient of 2.39 A(3) Da(-1) and 47% solvent content. The unit-cell parameters of the crystals were a = b = 123.2, c = 35.6 A. Microseeding and further optimization yielded crystals with an X-ray diffraction limit of 2.36 A. A single 70 degrees data set was collected and processed, resulting in an overall R(merge) and a completeness of 9.5% and 99.3%, respectively.
Hughes
Ronny C
RC
Oak Ridge National Laboratory, Neutron Scattering Science Division, 1 Bethel Valley Road, Oak Ridge, TN 37831, USA.
Tomanicek
Stephen J
SJ
Ng
Joseph D
JD
Coates
Leighton
L
D003460:Crystallization; D018360:Crystallography, X-Ray; D043223:Deoxyribonuclease IV (Phage T4-Induced) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004795:Enzyme Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D043223:Deoxyribonuclease IV (Phage T4-Induced)