Computational protocol: Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

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Protocol publication

[…] Generally, crystal hits were initially prepared in sitting drop 96-well format. Proteins were set up as 1 µL protein+1 µL reservoir solution and incubated at 18°C for 24 h to 1 mo. If crystal optimization was required it was performed in 24-well hanging drop format with 1 µL protein+1 µL reservoir solution. Crystals were cryoprotected with mother liquor with 10%–15% glycerol. Datasets were collected on an in-house FR-E SuperBright Cu rotating anode/Raxis IV++ detector (Rigaku Americas, The Woodlands, TX, USA); except for PPIC, which was collected at APS 19-BM. Data was integrated and scaled using the HKL2000 program package ,. The program PHASER was used as part of the CCP4 suite to find the molecular replacement solution. Manual rebuilding was performed using either O or COOT , and refined using REFMAC in the CCP4I program suite . In most cases ARP/wARP was utilized to assist in model building and iterative refinement of starting phases . Final models were evaluated using PROCHECK and MOLPROBITY , with all models judged to have excellent stereochemistry and no residues in disallowed regions of Ramachandran space. […]

Pipeline specifications

Software tools CCP4, Coot, ARP/wARP, PROCHECK, MolProbity
Application Protein structure analysis
Organisms Homo sapiens
Chemicals Cyclosporine