Computational protocol: Structural and Thermodynamic Basis of Epitope Binding by Neutralizing and Nonneutralizing Forms of the Anti-HIV-1 Antibody 4E10

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Protocol publication

[…] Diffraction data from a single crystal of the unbound Fab were collected using Beamline I02 of the Diamond Light Source (Oxfordshire, United Kingdom) under cryogenic conditions (100 K). Diffraction data of the WDWD and ΔLoop Fabs in complex with 4E10ep were collected in Beamline BL5A of the Photon Factory (Tsukuba, Japan) also under cryogenic conditions (100 K). Diffraction images were processed with the program MOSFLM and merged and scaled with the program SCALA of the CCP4 suite (). The structures were determined by the method of molecular replacement using the coordinates of recombinant WT Fab previously determined by us () with the program PHASER (). All models were refined with the program REFMAC5 () and manually built with COOT (). The crystal of unbound WT Fab was partially twinned (ratio 70/30) as determined with the program REFMAC5 during the refinement stage. Validation was carried out with PROCHECK (). Because of the weak electron density in the apex of the CDR-H3 (100WGWLG100D) in the crystal structure of unbound 4E10, additional validation steps were taken to rigorously evaluate the geometry of the residues modeled in this region. First, we verified no geometrical violations were found among the residues comprising the apex with the validation tools of CCP4 (), PHENIX (), and the PDB () with respect to bond lengths, bond angles, dihedral angles, chirality restrains, root mean square distances from planarity, psi-phi angles, rotamers, Cβ deviations, and geometrical clashes (including symmetry-related clashes). Second, we analyzed the top five automatic conformations of residues of the apex built by the program ARP-WARP (). Although these conformations were built automatically (no human intervention), they showed great similarity to the conformation built by us (root mean square deviation [RMSD]-Cα = 0.8 ± 0.2). Third, the program RINGER was used to look into the conformational variability of the side chains of residues WH100, WH100B, and LH100D of the apex (GH100A and GH100D were not analyzed because they do not possess side chains). The individual propensity plots of each residue indicate that the side chains adopt a unique conformation above the standard electron density threshold value of 0.3 σ () (see Fig. S1 in the supplemental material). Collectively, the exhaustive validation procedure described above demonstrates that, although the region of the apex of CDR-H3 of the unbound 4E10 is highly dynamic (a general feature of the CDR-H3 apex of 4E10; see Fig. S1b in the supplemental material), the conformation modeled has adequate geometry and fits reasonably well the otherwise weak electron density present in that region. (Data collection and structure refinement statistics are given in .) […]

Pipeline specifications

Software tools CCP4, REFMAC5, Coot, PROCHECK, PHENIX, ARP/wARP
Application Protein structure analysis
Organisms Human immunodeficiency virus 1
Diseases HIV Infections
Chemicals Tryptophan