Computational protocol: Unexpected structure for the N-terminal domain of hepatitis C virus envelope glycoprotein E1

Similar protocols

Protocol publication

[…] Crystals of nE1 were grown by sitting-drop vapour diffusion at 20.5 °C, in 96-well plates (Greiner Bio-One Ltd, Stonehouse, UK). Plates were set up with a Cartesian Technologies MIC4000 robot, as previously described.nE1 crystallized in 15% (wt/v) polyethylene glycol 1500, 3.6% (wt/v) polyethylene glycol 4000 and 0.05 M sodium acetate pH 4.8. Crystals were of either hexagonal or tetragonal morphology of which the latter gave better diffraction. Addition of 100 nl of 6–8% of 2,5-hexanediol or 1,6-hexanediol lead to larger crystals of approximate size 110 × 30 × 10 μm.Crystals were flash frozen into liquid nitrogen using 25% (v/v) ethylene glycol/resevoir solution as cryoprotectant. Although the crystals diffracted very weakly, data sets could be recorded from crystals at 100 K at Diamond Light Source (Didcot, UK, beamlines I24 and I04) and were processed with either HKL2000 (ref. ) or XIA2 (ref. ) in the space group P41212 with unit cell dimensions a=b=105.0 Å, c=204.8 and α=β=γ=90°. For the native data collection, numerous crystals were screened to eventually get a crystal diffracting to higher resolution (3.5 Å), whereas for the sulphur single-wavelength anomalous dispersion (Sulphur-SAD) data collection 32 crystals were used. Processing statistics are summarized in . [...] Experimental phasing with heavy atoms was unsuccessful and the absence of methionines prevented the use of selenomethionine for structure determination. Nonetheless, HCV E1 (residues 1–79) contains four cysteines, thus data sets from 32 crystals were collected at a wavelength of 1.77 Å at beamline I04 using inverse beam mode in 5° wedges and merged with XIA2 () to use sulphur-SAD phasing. HKL2MAP was used to locate sulphur sites using the merged data cut to 7 Å resolution (if cysteines are involved in disulphides, the anomalous signal decays rapidly beyond this point). Twelve sulphur sites (presumed to be the centroids of disulphide bonds) were input to PHENIX autosol giving electron density maps that were difficult to interpret; however, it was possible to place six α-helices in the map from which non-crystallographic symmetry (NCS) matrices could be extracted. Density modification using a solvent content of 75%, sixfold NCS averaging and extension of the resolution to the native data set (3.5 Å) gave interpretable maps. The location of the cysteines and glycans allowed the alignment of the sequence into density. Model building was performed with COOT and refinement with AUTOBUSTER, using sixfold NCS restraints automatically generated by the local structural symmetry restraints () The final Rwork/Rfree values of the model were 22/24% and geometry was checked with MolProbity. For refinement statistics see . Data collection and structure determination are described in greater details elsewhere. […]

Pipeline specifications

Software tools xia2, HKL2MAP, PHENIX, Coot, MolProbity
Application Protein structure analysis
Organisms Hepacivirus C, Classical swine fever virus
Diseases Hepatitis C