Computational protocol: Three-Dimensional Architecture of the Human BRCA1-A Histone Deubiquitinase Core Complex

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Protocol publication

[…] Particles were semi-automatically extracted in 120×120 pixel boxes using the EMAN2 boxer swarm tool () and followed by manual examination. The non-crosslinked dataset consisted of 1,592 particles, and the crosslinked dataset consisted of 8,393 particles. Images were filtered to the first zero of the contrast transfer function (22 Å). Reference-free 2D alignment, classification, and averaging of particle images were carried out using IMAGIC software (). Initial maps of the crosslinked complex assuming C2 symmetry were calculated using angular reconstitution and improved by anchor-set refinement. Deviations from an apparent D2 arrangement for the reconstructed volume were significant and application of higher symmetry was not justified. This map was masked and filtered to 45 Å and then subsequently refined using Frealign (v9.11) (), increasing the high-resolution limit of the data used for refinement in 5 Å steps to 30 Å. To validate the Fourier shell correlation (FSC) curve, the final round of refinement was repeated using image data with phases greater than 30 Å randomized (). The FSC between two maps calculated from different subsets of particle images provided a resolution estimate of 24.8 Å using the 0.143 threshold criterion (). A final refinement to 27 Å produced no further increase in resolution (B). Maps were displayed and the docking of atomic coordinates was performed with UCSF Chimera (). Segmented maps produced with the Segger-option were used for estimating the volume of the half map to approximate the non-crosslinked complex. Volumes for the UEV and VWA domains, blurred to 60 Å, were generated using pdb2mrc in EMAN. […]

Pipeline specifications

Software tools EMAN, IMAGIC, Frealign, UCSF Chimera, Segger
Application cryo-EM
Organisms Homo sapiens
Diseases Ataxia Telangiectasia, Neoplasms, Hereditary Breast and Ovarian Cancer Syndrome