Table 1

Data collection and refinement statistics. For crystallization, acidic and basic leucine zippers were removed from I-Ag7 by thrombin digestion, and the residual COOH-terminal spacer sequences were digested with carboxypeptidase B. Covalent I-Ag7–peptide complexes of GAD207-220 and GAD221-235 were concentrated to 10 mg ml−1 in 20 mM Hepes, 25 mM NaCl (pH 7.5) and screened for crystallization. Small crystals were obtained for I-Ag7–GAD207-220 and enlarged by streak seeding (42). Crystals were then grown in 16 to 18% polyethylene glycol 4000, 0.2 M LiCl (pH 6.6), 1% 2-methyl-2,4-pentanediol and flash-cooled to 96 K with glycerol as a cryoprotectant (through a sequential soak in 1, 5, 10, and 15% glycerol). A single crystal was used to collect data at beamline 7-1 of the SSRL. Data were integrated in space group C2221(a = 95.1 Å, b = 110.1 Å,c = 96.1 Å) with DENZO (version 1.9.1) and reduced with SCALEPACK [version 1.9.0 (43)]. Crystal mosaicity was refined in batches and ranged from 1.0 to 1.3°. A single I-Ag7 was assumed to be present in the asymmetric unit based on the calculated Matthew's coefficient [V m ∼ 2.9 Å3/Dalton, ∼57.0% solvent (44)]. Molecular replacement was carried out with CCP4's version of AMoRE (45,46) and the MHC class II search model I-Ak [PDB code 1iak (24)], in which the peptide, carbohydrate, and waters had been omitted. Clear rotation (one peak greater than 50% maximum peak height, resolution 8.0 to 3.5 Å, sphere radius 22.5 Å) and translation solutions (correct solution was 6.7σ above the next highest peak, resolution 8.0 to 3.5 Å) were found. After rigid body fitting within AMoRE (resolution 20.0 to 2.7 Å), the correlation coefficient was 68.7% and theR cryst was 41.5%. Further rigid body refinement was carried out with CNS [version 0.9 (47)] by using three domains α11, α2, and β2. TheB value for all atoms was then reset to 20 A2. Cross-validated σA-weighted 2F oF c andF oF celectron density maps (48) were used throughout in order to rebuild and were viewed with the graphics program O (49). Density was observed for the peptide at this stage, but not refined until later. Because density for the helix region Gly β54 to Gln β64 was ambiguous, its occupancy was set to zero for the next refinement cycle (positional and torsion angle dynamics refinement, resolution 10.0 to 2.7 Å, with a starting temperature for the dynamics determined with the slow cool protocol of 4000 K). Density for the helix was noticeably improved and was gradually rebuilt over the next few cycles. R free (50) (∼10% of the reflections) was used from the outset to monitor the course of the refinement. Waters were picked toward the end of the refinement, with an F oF c map and a 3.5 to 4.0σ cutoff. Waters were rejected after refinement if they failed to appear in both 2F oF c andF oF cmaps, were not within hydrogen bonding distance of another protein atom or water molecule, or if their B value exceeded 50 A2. All data from 40 to 2.6 Å were used in the last few refinement cycles, and an anisotropic B correction was applied along with a bulk solvent correction to give the final structure (51).

Data collection
Resolution range (Å)40.0–2.6
Unique reflections15,754 (1545)*
Total number of reflections66,107 (6531)
Completeness (%)99.3 (99.6)
I/σ(I)〉15.7 (4.2)
Rsym (%)6.8 (35.3)
Refinement statistics
Resolution range (Å)40.0–2.6
Anisotropic correction (Å2)B11 = 8.0,B22 = −3.5, B33 = −4.5
R cryst 0.210 (0.276)
R free § 0.258 (0.344)
rmsd from ideality
Bond length (Å)0.006
Bond angle (°)1.3
Dihedrals (°)26.0
Impropers (°)0.8
I-Ag7 36.4
Peptide GAD207–220 35.4
Ramachandran plot|
Favord (%)90.2
Allowed (%)9.2
Generous (%)0.0
Disallowed (%)0.6
  • * Data for the highest resolution shell (2.69 to 2.60 Å).

  • Rsym = 100 × [ΣhΣi|Ii(h) − 〈I(h)〉|/ΣhI(h)], where Ii (h) is theith measurement of the h reflection and 〈I(h)〉 is the average value of the reflection's intensity.

  • Rcryst = Σ∥Fo|−|Fc∥/Σ|Fo|, where Fo andFc are the observed and calculated structure factor amplitudes within the set of reflections used for refinement.

  • § Rfree = Σ∥Fo| − |Fc∥/Σ|Fo| calculated for a randomly selected set of structure factors (∼10%) throughout the resolution range and not used in refinement.

  • | The Ramachandran plot was generated with PROCHECK (52).