Table 1

Crystallographic analysis. Crystals of the ternary complex were grown by the conventional hanging-droplet vapor diffusion method at room temperature. The Escherichia coli–expressed scD10 (57) and glycosylated CA/I-Ak from CHO Lec3.2.8.1 cells (58) were mixed at a 1:1 molar ratio to a final concentration of 23 mg/ml in 0.1 M tris-HCl buffer (pH 8.5). The protein solution was further mixed with a crystallization buffer of 8% PEG 8000, 0.1 M tris (pH 8.5), 0.01 M KCl and then sealed against a reservoir with the same buffer. These crystals belong to the space groupP21212 with unit cell parametersa = 97.6 Å, b = 345.3 Å, and c= 97.7 Å. There are two complexes in the asymmetric unit with 78% solvent. Crystals were stepwise transferred to cryoprotectant solution that contains 30% glycerol in addition to the crystallization buffer before freezing. One data set was collected at the SBC-CAT of Advanced Photon Source (APS) at the Argonne National Laboratory with an APS1 mosaic 3 × 3 CCD detector at 100 K. The wavelength used was 1.069 Å. Data were processed with programs DENZO and SCALEPACK (59). The structure was solved by molecular replacement with AMoRe (60). The refined structure of CA/I-Ak(61) was taken as the search model. At the beginning, only one of the CA/I-Ak pMHC molecules (molecule A) was identified. The CA/I-Ak molecule B was located only after the first one was rigid body refined and fixed. We then carried out the rigid body refinement of the two I-Ak molecules, each of the domains and the bound peptide being treated as one rigid body. A few degrees of rotations were seen for the α2 and β2 domains. After positional and individual B-factor refinement, the R free dropped, and the Ig-like domains of scD10, especially the one in complex A, were already visible in the calculated 2F o F c difference map. Cycles of model building and refinement gradually improved the density, allowing the correct side-chain assignment and eventually the completion of the model building and refinement. All the refinement was done with the program X-PLOR (62) and model building with program O (55). Ten percent of reflections were set aside forR free calculation. Noncrystallographic symmetry (NCS) refinement was not used because the two complexes are related by an improper rotation and the two I-Ak molecules have very different temperature factors. Using NCS at an early stage of the refinement led to serious intermolecular collision. In the current model, each of the complexes contains residues 1 to 182 and 2 to 190 of I-Ak α and β chains, respectively, all 16 residues of the bound peptide (three leader-derived and 13 CA-derived), as well as residues 2 to 117 and 3 to 116A of D10 Vα and Vβ domains, respectively. Ten carbohydrate moieties were modeled in three potential glycosylation sites in I-Ak molecules. At this resolution no water molecules were included. The final 2F oF c map is of excellent quality, particularly in the TCR regions and the interface between TCR and pMHC. There are very few density breaks, mainly in the BC loops of the I-Ak β2 domains. Figure 2 is an omit map in the bound peptide region.

Data collection
Resolution limit (last shell)30.0 to 3.2 Å (3.31 to 3.20 Å)
Total number 501,406
Unique 52,592 (I > 0)
I/σ(I) 10.0 (2.2)
Completeness95.2% (87.1%)
R merge * 7.0% (29.2%)
Refinement statistics
Resolution range15.0 to 3.20 Å
Number of reflections 46,332 (F > 0)
R work 24.7%
R free 29.3%
rms deviations
Bonds 0.007 Å
Angles 1.4°
Dihedrals 30.2°
Ramachandran plot
Favored 71.4%
Generous 6.9%
Unfavored 0%
  • * Rmerge = Σ(|Ii(hkl) − 〈Ii(hkl)〉|)/ΣIi(hkl).

  • In working set.