Computational protocol: In vivo epidermal migration requires focal adhesion targeting of ACF7

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Protocol publication

[…] Crystals of ACF7-NT were obtained using hanging-drop vapour diffusion at 4 °C. For ACF7-NT, a 1 μl 20 mg ml−1 ACF7-NT in a buffer containing 25 mM Hepes pH 7.5, 150 mM NaCl, 2 mM CaCl2 was mixed with 1 μl of reservoir solution containing 0.1 M MES pH 6.5, 0.2 M (NH4)2SO4 and 1 M (NH4)2HPO4. Cryoprotection was achieved by raising the glycerol concentration stepwise to 30% with a 5% increment in each step. The diffraction data sets of ACF7-NT were collected at 100 K at Shanghai Synchrotron Radiation Facility. Diffraction data were processed with DENZO and scaled with SCALEPACK. The ACF7-NT structure was determined by molecular replacement with Phaser in CCP4i (ref. ) using known structures (PDB code=3f7p and 2odv, respectively) as the search model. After tracing the initial model manually in the program Coot, the model was then refined against the native data at 2.6-Å resolution using CNS and PHENIX. The structure data have been deposited to Protein Data Bank (PDB) with accession code 4Z6G. [...] SAXS data for ACF7-NT and ACF7-NT (Y259D) in PBS were collected at room temperature at 12ID-B, APS, ANL using 1 mg ml−1 protein and an incident X-ray wavelength of 0.886 Å. The data was reduced and analysed using ATSAS. PRIMUS was used to determine the Rg value in reciprocal space. The SAXS data was deposited to SASBDB with accession code SASDBL3 for ACF7-NT and SASDBN3 for ACF7-NT Y259D. […]

Pipeline specifications

Software tools Coot, CNS, PHENIX, ATSAS
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Chemicals Tyrosine