Computational protocol: Structure of a clade C HIV-1 gp120 bound to CD4 and CD4-induced antibody reveals anti-CD4 polyreactivity

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Protocol publication

[…] We collected a complete data set to 2.2 Å resolution from cryoprotected 21c Fab crystals at beamline BL12-2 of the Stanford Synchrotron Radiation Lightsource (SSRL) on a MarMosaic-325 CCD detector and processed the data using HKL2000. We found a molecular replacement solution using Phaser utilizing two portions of a Fab (VH-VL and CH1-CL; PDB code 3C08 as search models. We refined the model using TLS-restrained refinement implemented in Refmac and used Coot for model building. The final model (Rwork=19.2%; Rfree=21.8%) includes 3262 protein atoms, 181 water molecules and two 2-methyl-2,4-pentanediol molecules (). 97%, 2.5% and 0.5% of the residues were in the favored, allowed, and disallowed regions, respectively, of the Ramachandran plot. Five residues at the C-terminus of the heavy chain and two residues at the C-terminus of the light chain were disordered.We collected data to 3.4 Å (~3.9 Å in the direction of b/b*) from cryoprotected crystals of the CAP210–sCD4–21c complex and processed the data without a B-factor correction using MOSFLM and Scala. The crystals usually exhibited weak, highly mosaic and anisotropic diffraction. We solved the structure by molecular replacement using Phaser by searching for four components; VH-VL and CH1-CL from the 21c Fab structure, the HXBc2 core plus CD4 D1 (PDB code 2NXY, and CD4 D2. We performed ellipsoid truncation and anisotropic scaling using the Diffraction Anisotropy Server, resulting in greatly improved electron density maps and data that were 79% complete to 3.4 Å. We refined the model using CNS,, group B factors, and composite omit maps to Rwork=23.0%; Rfree=32.1% (Rfree based on 5% of the reflections). To finalize the model we performed an additional refinement step using a reduced set of reflections for calculating Rfree (0.5%) in a maximum log likelihood function refinement.The final model includes 6894 total protein atoms (2272 atoms of CAP210, 1362 atoms of sCD4, and 3260 atoms of Fab 21c), and 56 carbohydrate atoms in four ordered N-acetylglucosamine molecules (attached to gp120 Asn441, Asn380, Asn272 and Asn258). Some residues (mainly surface-exposed) were missing portions of sidechain electron density. We assigned potential aspartic acid residues at N-linked glycosylation sites (formed from asparagines by treatment with PNGase F) as asparagines. The electron density at some solvent-exposed regions was poor, but was higher quality at protein interfaces (). The 21c Fab heavy chain portion of the model includes the same number of residues as in the free 21c structure, and the light chain includes all but the N- and C-terminal residues. sCD4 is missing one residue at the N-terminus and 16 residues at the C-terminus including the 6x-His tag. Disordered residues in the CAP210 structure are indicated in Supplementary . 97.0%, 1.3% and 1.7% of the residues were in the favored, allowed, and disallowed regions, respectively, of the Ramachandran plot.We used AreaIMol in CCP4 and a 1.4 Å probe for calculated buried surface areas and PyMol for superposition calculations and molecular representations. […]

Pipeline specifications

Software tools Coot, CCP4, CNS, PyMOL
Application Protein structure analysis
Organisms Human immunodeficiency virus 1, Viruses
Diseases HIV Infections