Protein expression and purification. The BRC4 - RAD51 fusion construct was subcloned into pGAT3, a member of the pGAT series of expression vectors that allow production of the target gene fused to a double amino-terminal tag consisting of a six histidine sequence followed by the glutathione-S-transferase protein 35. The BRC4 - RAD51 fusion protein was overexpressed in E. coli strain BL21(DE3) for three hours at 37°C by addition of 0.2 mM IPTG. The soluble protein was recovered from the crude bacterial lysate by Ni-NTA agarose chromatography (QIAGEN). The tag was cleaved by incubation with the TEV NIa protease and selectively removed by glutathione agarose chromatography (Amersham Biosciences). The protein was purified to homogeneity by two further steps of anion exchange chromatography on a RESOURCE Q 6ml column (Amersham Biosciences) and gel filtration on a Superdex 200 HR 10.30 column (Amersham Biosciences). The purified protein was concentrated to 12 mg/ml (0.38 mM) in 20 mM Hepes pH=7.2, 100 mM NaCl, 1 mM DTT, flash frozen in liquid nitrogen and stored in aliquots at –80° C.
Figure 6 The RAD51 ATP-binding site. a Examination of the nucleotide-binding pocket of BRCA2-bound RAD51 shows that residues critical for ATP binding and hydrolysis, such as Thr133, Lys134 in Walker motif A and Asp222, in Walker motif B, are sequestered in a solvent-inaccessible hydrogen-bonding network that extends to Tyr159, Asp161 and Thr165 via a buried water molecule. Side chains of residues important for ATP catalysis, together with adjacent, interacting amino acids, are shown as sticks. A green sphere indicated the position of a buried water molecule. Dashed yellow lines represent hydrogen bonds. b A 3-D superposition shows that in BRCA2-bound RAD51 the ATP-binding loop (cyan) adopts a more closed conformation relative to the ADP-bound form of RecA (green), which is likely to preclude its occupation by the ATP phosphates. The atoms of the ADP molecule are drawn as spheres of Van der Waals radii.
Table 1 Crystallographic data on the RAD51-BRC4 complex.
Diffraction data (space group: P212121: a=57.30Å, b=59.14Å, c=77.20Å)Dataset / Resolution / Wavelength / Reflections1 (unique) / Completeness (outer shell) / Rsym2 (outer shell) / I/(I) / Beamline
Native / 1.8Å / 1.5418Å / 169388 (24702) / 99.9 (99.1) / 0.051 (0.308) / 40.9 (6.7) / In-house
KAu(CN)2 / 2.0Å / 1.5418Å / 179758 (18077) / 100.0 (100.0) / 0.059 (0.194) / 36.6 (11.9) / In-house
SeMet, peak / 1.7Å / 0.9792Å / 204230 (29143) / 99.9 (99.9) / 0.077 (0.321) / 23.5 (6.5) / ESRF, ID-29
SeMet, remote / 1.7Å / 0.90831Å / 207259 (29329) / 99.9 (99.6) / 0.070 (0.481) / 24.7 (4.2) / ESRF, ID-29
PhasingKAu(CN)2 / SeMet, peak / SeMet, remote
Rcullis (iso/ano)3 / 0.93 / 0.95 / - , 0.70 / 0.84 / 0.84
Phasing power (iso/ano)4 / 0.72 / 0.74 / - , 2.1 / 0.48 / 1.65
Figure of merit5 / 0.21 / 0.51
Refinement6Resolution (Å) / Reflections / Number of non-H atoms / R7 (%) / Rfree (%) / <B> (A2) / Rmsd bonds (Å) / Rmsd angles (°)
24.8-1.7 / 55746 / 2179 / 19.1 / 20.6 / 21.1 / 0.006 / 1.229
1 For MAD data, the Bijvoet pairs were not merged.
2 Rsym =
3 Rcullis as defined in SHARP.
4 Phasing power as defined in SHARP.
5 Figure of merit as defined in SHARP.
6 Statistics for all data.
7 R-factor =