Computational protocol: Endocytic sorting motif interactions involved in Nef-mediated downmodulation of CD4 and CD3

Similar protocols

Protocol publication

[…] Native diffraction data sets of all crystals were recorded at cryogenic temperature of 100 K using a PILATUS 6 M detector on beamline X10SA (PXIII) at the Swiss Light Source (SLS, Villigen, Switzerland). The data was processed, integrated, and scaled using the XDS package. Data collection and refinement statistics are summarized in Table .The space group of the Nef-B–HckSH3-E crystal was identified as P32 with unit cell dimensions a = b = 104.0, c = 53.0. The asymmetric unit contained two copies of Nef-B–HckSH3-E. The structure was solved by molecular replacement with phaser using the previously published SIV Nef structure as the search model (PDB: 3IK5). After initial simulated annealing (torsion angle) in phenix.refine, the model was refined to a resolution of 2.78 Å by multiple iterations of alternating manual model rebuilding in COOT and positional refinement with phenix.refine using non-crystallographic symmetry restraints.The crystal grown of Nef-E–CD4LL1 diffracted up to 2.50 Å. The space group was identified as C2221 with unit cell dimensions of a = 51.48, b = 140.73, c = 106.43, which gave enough space for two Nef-E molecules. A 1.35 Å SIV Nef-core structure, which was previously solved in our group resolving SIVmac239 Nef residues 103–183, 204–233 was used as a search model in PHASER (CCP4 package) to solve the Nef-E–CD4LL1 structure with molecular replacement. Alternating cycles of manual building in COOT and refinement in REFMAC5 and Phenix were performed to generate the final structural model. Data collection statistics and refinement parameters for both structures are shown in Table . Molecular diagrams were created using PyMOL (Schrödinger, MA). The electrostatic surface potential was calculated with adaptive Poisson-Boltzmann solver (APBS). […]

Pipeline specifications

Software tools XDS, PHENIX, Coot, CCP4, REFMAC5, PyMOL
Applications Small-angle scattering, Protein structure analysis
Chemicals Tyrosine