Table 1

Crystallographic statistics for the complex between vIL-6 and gp130. KSHV vIL-6 in complex with the D1D2D3 domains of human gp130 was prepared by coinfection of sf9 cells with recombinant baculovirus secretion constructs of the individual proteins (28). Nonglycosylated vIL-6 and gp130 were produced by carrying out the expression in the presence of tunicamycin, an inhibitor of N-linked glycosylation (29). The secreted complexes were harvested from the supernatant and purified by Ni-NTA, gel filtration, and anion exchange chromatography (28). The nonglycosylated complex (10 mg/ml) crystallized from 10% MPEG-2K and 0.1 M Na citrate (pH 6.5) in space groupC 2, with unit cell dimensions ofa = 103 Å, b = 123.31 Å,c = 76.79 Å, β = 112.03°. A Matthews coefficient calculation (4.2 Å3/dalton) indicated a 70% solvent content for one vIL-6 and one gp130 in the asymmetric unit (45). Therefore, a half-tetramer (or half-dimer) is present in the asymmetric unit, related by C 2 symmetry to the other half of the tetramer. The crystals were flash-frozen in liquid nitrogen in the presence of mother liquor containing 20% ethylene glycol, and data sets were collected at ALS beamline 5.02 and SSRL 9-2. A single data set from a crystal that diffracted to beyond 2.4 Å was collected, and integrated and scaled using MOSFLM and SCALA, respectively (45). Initial phases for two domains of gp130 were obtained by molecular replacement with AmoRe (45) using the coordinates of the CHR (D2D3) domain of gp130 (PDB ID: 1BQU) (34). For vIL-6, only MOLREP (45) succeeded in finding a molecular replacement solution using the coordinates of huIL-6 (PDB ID: 1ALU) (37) in which the residues were truncated to alanine. No model of an Ig domain gave a satisfactory solution for the gp130 D1 domain, so the structure of the D1 domain was traced from the electron density calculated with the partial structure of the refined D2D3 and vIL-6. The entire structure was then built using XFIT, as implemented in XTALVIEW (45). The structure was refined against a maximum likelihood target function, and consisted of a rigid-body refinement, several cycles of simulated annealing with torsion angle molecular dynamics, and iterations between positional andB-factor minimization. The structure was manually rebuilt using SIGMAA-weighted 2F obsF calc and omit F obsF calc maps as implemented in CNS and the graphics program O (45), and stereochemical analysis was performed with PROCHECK (45). All data between 50 Å and 2.4 Å were used, except for 5% data randomly selected for cross-validation. All residues of the receptor (2 to 302) and the body of the cytokine (6 to 172) are ordered in the electron density (residues 2 to 6 and 173 to 180 are not visible in the vIL-6 electron density). The A/B loop of vIL-6, which is often disordered in uncomplexed cytokine crystal structures, is entirely ordered and exhibits average temperature factors. No evidence of N-linked glycosylation is present on either the cytokine or the receptor.

Data collection
Resolution (Å) (highest resolution shell)50.0 to 2.4 (2.53 to 2.40)
Measured reflections106,397
Unique reflections34,376
Completeness (%)99.0 (99.6)
Rmerge (%)*8.1 (48.7)
I/σ(I)3.4 (1.4)
Mosaicity (°)0.4
Model refinement
Resolution range (Å)50.0 to 2.4 (2.53 to 2.40)
Rcryst (%)21.3 (31.3)
Rfree (%)§25.6 (34.6)
No. of protein atoms3783
No. of waters370
Average B factor (Å2)
Protein main chain46.3
Protein side chain49.8
Water54.9
RMSD angles (°) 0.007
RMSD bonds (Å) 1.6
Ramachandran plot (%)
Most favored87.9
Allowed12.1
  • * Rmerge = ΣhklI – 〈I〉∣/ΣhklI, whereI is the intensity of unique reflection hkl, and 〈I〉 is the average over symmetry-related observation of unique reflection hkl.

  • The RMSD in bond lengths and angles is the RMSD from the ideal stereochemical values.

  • Rcryst = Σ∣FobsFcalc∣/ΣFobs, where Fobs and Fcalc are the observed and calculated structure factors, respectively.

  • § Rfree is Rusing a set of reflections sequestered before refinement.