Computational protocol: Structural insights into the interaction of the ribosomal P stalk protein P2 with a type II ribosome-inactivating protein ricin

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Protocol publication

[…] Crystals of the chimeric protein were grown using the hanging-drop vapour-diffusion method at 289 K and yielded crystals in two days when using a well solution that contained 0.4 M ammonium dihydrogen phosphate. For data collection, all crystals were soaked in a cryoprotectant solution consisting of the respective reservoir solution supplemented with 20% (v/v) glycerol, and they were then flash-frozen in liquid nitrogen. Data sets for all crystals were collected on beamline 17U at the Shanghai Synchrotron Radiation Facility (SSRF) at 100 K. The data were processed and scaled using the HKL2000 package and programmes in the CCP4 package. Statistical parameters for the diffraction data are summarised in . [...] The structure of the chimeric protein was determined by molecular replacement using MOLREP, as implemented in the CCP4 suite. The structure of ricin A-chain (Protein Data Bank code 2AAI) was used as the search model. An initial model containing the ricin A-chain was obtained and refined using the maximum likelihood method, as implemented in REFMAC5. The C10-P2 peptide was manually rebuilt in Coot. The final model was evaluated using the programmes MOLPROBITY and PROCHECK . Sequence alignment was performed using the programme ClustalW . The crystallographic parameters are listed in . All of the structures shown in the figures were prepared using PyMOL (DeLano Scientific). […]

Pipeline specifications

Software tools CCP4, Molrep, REFMAC5, Coot, MolProbity, PROCHECK, Clustal W, PyMOL
Application Protein structure analysis
Organisms Homo sapiens
Chemicals Hydrogen