Computational protocol: The p97-FAF1 Protein Complex Reveals a Common Mode of p97 Adaptor Binding*

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Protocol publication

[…] To prepare the cryo-EM grids, 5 μl of purified p97-FAF1 at 0.2 mg/ml were applied to glow-discharged holey-carbon grids, which were then blotted and flash frozen in liquid ethane using a Vitrobol (FEI). Data were collected on a CM200 (Philips) electron microscope, operating at 200 kV, under low dose conditions, at 50,000× magnification, and at 2–4 μm nominal defocus. For the nanogold labeling, a 0.05 mg/ml complex of C-terminally His-tagged FAF1 and untagged p97 was mixed with 5 nm of nickel-nitrilotriacetic acid nanogold. The sample was applied to glow-discharged continuous-carbon grids and stained with 2% uranyl acetate solution. Images were collected using a Teitz camera at the Imperial College Electron Microscopy Centre.Data processing was carried out using Imagic (version 5) (), unless stated otherwise. The micrographs were coarsened by a factor of 2, leaving a pixel size of 3.52 Å/pixel, and contrast transfer function-corrected. 60,032 particles were picked interactively using BOXER in EMAN, and band-pass filtered between 10 and 200 Å and then normalized and masked. For the p97-FAF1 data set, reference-free class averages were created from successive rounds of multivariate statistical analysis and alignment (brute force alignment (). For the four-dimensional data processing, multiple side view class averages were used to make initial three-dimensional models with C6 symmetry. These were refined by successive rounds of competitive alignment and projection matching to separate the data set. Models representative of p97-FAF1 complex were judged visually, and the particles that aligned to them were extracted and processed separately. A single side view was selected and used to make an initial 6-fold model that was refined without symmetry by progressive rounds of alignment and projection matching. The process was then repeated with 3-fold symmetry imposed throughout the processing. The final C3 p97-FAF1 protein complex model has been deposited on the EMDB website (accession code EMD-2319). […]

Pipeline specifications

Software tools IMAGIC, EMAN
Application cryo-EM
Organisms Homo sapiens
Diseases Neoplasms