Computational protocol: Structures of Falcipain-2 and Falcipain-3 Bound to Small Molecule Inhibitors: Implications for Substrate Specificity‡

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Protocol publication

[…] Crystals were cryoprotected in mother liquor supplemented with 25% (FP2−E64) and 15% ethylene glycol (FP3−leupeptin), respectively. Samples were then mounted in standard Hampton cryoloops, flash-cooled in liquid nitrogen, and loaded into a Stanford automated mounting system (SAM) sample cassette.() Data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) beamline 9-1, Menlo Park, CA, on an ADSC Q-315 detector.Diffraction data were collected from a single FP2−E64 crystal as 0.5°, 35 s oscillations. FP2−E64 crystals belong to space group C2221 (a = 143.78 Å, b = 167.81 Å, c = 177.76 Å). Diffraction data from a single FP3−leupeptin crystal were collected as 1°, 20 s oscillations. FP3 crystals belong to space group R32 (a = b = 154.57 Å, c = 129.01 Å, β = 120°). Reflections were indexed and integrated using XDS() for FP2−E64 and MOSFLM for FP3−leupeptin.() Data were scaled and merged with XSCALE for FP2−E64() and SCALA for the FP3−leupeptin complex.() Because of the high redundancy of the data, we have included the precision indicating merging R-factor, Rpim() (calculated in SCALA), in Table as a more accurate description of the precision of the averaged measurements. Structure factor amplitudes were calculated using TRUNCATE.()The structure of FP2−E64 was determined by molecular replacement in PHASER() using the FP2 component of the FP2−cystatin complex (PDB 1YVB).() Four independent monomers were located in the asymmetric unit, yielding a solution with a log likelihood gain (LLG) of 4982 and translation function Z-score of 50.2. Following initial refinement in CNS,() mFo − DFc SIGMAA-weighted electron density maps confirmed that hemoglobin was absent. However, the presence of E64 was clear and we were able to position the small molecule inhibitor in the active site of all four monomers. The model was refined over several rounds in CNS, interspersed with manual adjustments in COOT.() Refinement was concluded in REFMAC5 using TLS parametrization.() The model shows good stereochemistry, as assessed by MOLPROBITY,() and is refined to a final Rfree and R-factor of 32.5% and 27.5%, respectively. Structure statistics (PDB 3BPF) are summarized in Table .The structure of FP3 was determined by molecular replacement in PHASER using one molecule of the FP2−E64 complex. Two monomers were located in the asymmetric unit, yielding a solution with a log likelihood gain (LLG) of 3661 and translation function Z-score of 59.9. Refinement/rebuilding of the model and placement of ligands followed the same protocol as the FP2−E64 structure. The model shows good stereochemistry, as assessed by MOLPROBITY, and is refined to a final Rfree and R-factor of 22.4% and 19.0%, respectively. Structure statistics (PDB 3BPM) are summarized in Table . […]

Pipeline specifications

Software tools CCP4, CNS, Coot, REFMAC5, MolProbity
Application Protein structure analysis
Organisms Plasmodium falciparum, Homo sapiens
Diseases Malaria
Chemicals Cysteine