Computational protocol: Crystal structure reveals vaccine elicited bactericidal human antibody targeting a conserved epitope on meningococcal fHbp

Similar protocols

Protocol publication

[…] The purified Fab (7.1 mg ml−1) was screened against 96 crystallization reagents (Index Screen, Hampton Research). Crystals were observed in multiple conditions; after 4 days, the largest crystals were found in 60% Tacsimate, pH 7.0. Optimization of the crystallization conditions was achieved in 57.5% Tacsimate (Hampton Research) at pH 6.0. Crystals were soaked in 15% PEG 3350 and 12% Tacsimate as cryoprotectant and cryo-cooled in liquid nitrogen. The crystals were tested for diffraction at beamline 5.0.1 at the Advanced Light Source, Lawrence Berkeley National Laboratory and several data sets were collected on an ADSC Q315R detector (Table ). Data were integrated and scaled with iMosflm. The structure of the Fab was solved using Phaser with a homology model constructed from the 1A12 sequence and the coordinates of the human anti HIV 21c Fab (PDB ID 3LMJ) using Swiss-Model.The Fab 1A12-fHbp complex at 10 mg ml−1 was screened with a matrix of ~ 800 crystallization conditions using a Crystal Gryphon robot (Art Robbins Instruments). Long plate-needle-looking crystals were found at days 7–10 with 17% PEG 3350 and 0.2 M sodium malonate (pH 4.0). Optimization of the original conditions was performed by changing the concentrations of the different components in the initial crystallization mixture. Changes in the pH did not yield any improvement. Crystals were soaked in the original mother liquor supplemented with 10 % ethylene glycol as cryoprotectant and prior to cryo-cooling in liquid nitrogen. Diffraction of the crystals was tested at beamline ID29 of the European Synchrotron Radiation Facility and several full data sets were collected on a Pilatus 6M detector. Diffraction data sets were indexed, integrated, and scaled with XDS and Aimless, via the CCP4 suite. The structure of the complex was solved by molecular replacement with Phaser using as model templates for fHbp, light chain, and heavy chains the coordinates deposited in the protein data bank under the codes 2YPV, 4YPG, and 4JQI, respectively. The CDR3 loop of the heavy chain was omitted in the template. [...] Initial molecular replacement solutions were subjected to subsequent cycles of manual building in Coot and refinement with Phenix.refine. The buried surface areas and atomic interactions/contacts, and the root mean square displacements, were calculated with PISA and Superpose, respectively. All the structural figures were created with PyMOL. […]

Pipeline specifications

Software tools iMosflm, SWISS-MODEL, XDS, CCP4, Coot, PHENIX, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens, Neisseria meningitidis