Download Figure 6 The RAD51 ATP-binding site

V07047A_supplementary_information
Methods
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 glutathioneS-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
Native
Wavelength
1.8Å
1.5418Å
Reflections1
Completeness
Rsym2
(unique)
(outer shell)
(outer shell)
169388
99.9 (99.1)
0.051 (0.308)
(24702)
KAu(CN)2
2.0Å
1.5418Å
I/(I)
Beamline
40.9
In-house
(6.7)
179758
100.0 (100.0)
0.059 (0.194)
(18077)
36.6
In-house
(11.9
)
1.7Å
SeMet,
0.9792Å
204230
peak
99.9 (99.9)
0.077 (0.321)
(29143)
1.7Å
SeMet,
0.90831Å
207259
remote
99.9 (99.6)
0.070 (0.481)
(29329)
23.5
ESRF, ID-
(6.5)
29
24.7
ESRF, ID-
(4.2)
29
Phasing
KAu(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
Refinement6
Resolution
Reflections
(Å)
24.8-1.7
Number of
R7
Rfree
<B>
Rmsd bonds
Rmsd angles
non-H atoms
(%)
(%)
(A2)
(Å)
(°)
2179
19.1
20.6
21.1
0.006
1.229
55746
1
For MAD data, the Bijvoet pairs were not merged.
2
Rsym =
  I (hkl)  I hkl   I hkl
i
hkl
i
i
i
hkl
i
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 =
F
obs
hkl
 Fcalc
F
obs
hkl