Computational protocol: The crystal structures of macrophage migration inhibitory factor from Plasmodium falciparum and Plasmodium berghei

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Protocol publication

[…] Data for PfMIF and PbMIF were collected on beamlines ID14-2 and ID14-4, respectively, at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) and were processed using the DENZO/SCALEPACK and CCP4 packages. [...] The structure proved difficult to solve by molecular replacement (MR) with the P21 data with calculations producing solutions that were inconclusive. All of the MR programs tested aligned the trimeric three-fold axis correctly but the rotation about this axis was very uncertain (i.e., the translation function was good but the subsequent rotation function was poor). In addition to the three-fold symmetry axis in the trimer, each subunit has pseudo two-fold symmetry. Self-rotation shows the crystallographic two-fold to be parallel to the molecular three-fold, and perpendicular to the pseudo two-fold axes, generating pseudo 222 symmetry. Additionally, the Patterson map showed noncrystallographic translation with a peak that was 43% of the height of the origin peak at 0.50, 0.45, and 0.00. This suggested that there were six molecules in the asymmetric unit correlating with the Matthews coefficient of 2.24 Å3 Da−1 (relating to a solvent content of 45%). All of this may have contributed to the difficulty in finding an MR solution for the P21 data.A partial solution was found using the automated molecular replacement pipeline Balbes following reprocessing of the data in the space group P1. Sixteen monomers were found using chain-A of coordinate set 2OS5 as the MR model. Of these, four protomers, related in pairs by 120°, satisfied the symmetry operations for P21 with the expected pseudo translation. The third chain of each trimer was generated and the subsequent model was good enough to start refinement. ARP/wARP was used to build in the P. berghei sequence, refinement was performed with REFMAC, and Coot was used to finalize the model. [...] Data collected for PfMIF were processed in the cubic space group P213. An ice ring forced the exclusion of some data during processing. Initial molecular replacement calculations using the human MIF coordinate set (pdb entry 1GD0) as the search model were unsuccessful most likely due to the low-sequence identity between PfMIF and the human homologue (∼29%). A solution was obtained when the PbMIF structure, which had not been available initially, was used as the model for molecular replacement with MolRep. Two protomers in the asymmetric unit were sought based on the Matthews coefficient (1.73 Å3 Da−1 with 29% solvent content) with the biologically relevant trimers being generated via the crystallographic three-fold axis. Data and refinement statistics for both structures are presented in Table . PDB accession codes are 2WKB and 2WKF for PbMIF and PfMIF, respectively. […]

Pipeline specifications

Software tools CCP4, ARP/wARP, Coot, Molrep
Application Protein structure analysis
Organisms Plasmodium falciparum, Plasmodium berghei, Homo sapiens
Diseases Malaria
Chemicals Mercaptoethanol