Structural Analysis of BRCA1-BARD1 Complexes

Samples of isolated wild-type, mutated, and modified BRCA1-BARD1 assemblies [0.02 mg/ml in 20 mM Hepes buffer (pH 7.2), 150 mM NaCl, 10 mM CaCl₂, 10 mM MgCl₂] were applied to glow-discharged, continuous carbon support films on copper grids (Ted Pella) or to EM affinity grids (24 (link), 38 (link)). Affinity grids were decorated with antibodies against the BRCA1 RING domain (EMD Millipore; MS110, AB1) or the BRCT domain (C-20) for labeling studies conducted on wild-type assemblies. Protein complexes were tethered to the antibody-decorated grids by incubating Ni-NTA eluates for 2 min, followed by standard negative staining procedures using 1% uranyl formate (39 (link)). Specimens were examined using a FEI TEM (FEI Company) equipped with a LaB6 filament and operating at 120 kV under low-dose conditions (<5 electrons/Ų). Images were recorded using an Eagle 2k HS CCD camera (FEI Company) with a pixel size of 30 μm at a magnification of about ×68,000 for a final sampling of 4.4 Å/pixel.