Similar protocols

Protocol publication

[…] Crystals of the CD1b–GMM complex were grown by the hanging-drop, vapour-diffusion method at 20 °C with a protein/reservoir drop ratio of 1:1, at a concentration of 10 mg ml−1 in 10 mM Tris–HCl pH 8, 150 mM NaCl using 20–30% PEG 4 K, 0.2 M Na Iodide and 2% ethylene glycol. Crystals of the GEM42 TCR in complex with the CD1b-Ile160Ala-GMM were obtained at a concentration of 2 mg ml−1 using 1.5–2 M NH4SO4, 0.1 M Tris–HCl pH8.5, 10 mM MgCl2 and 10 mM CeCl2.Crystals were soaked in a cryoprotectant solution containing mother liquor solution with PEG4000 concentration increased to 30% (v/v) for the CD1b–GMM crystals and 20% ethylene glycol for the ternary complex crystals, and flash frozen in liquid nitrogen. Data were collected (at 100 K) on the MX2 beamline at the Australian Synchrotron, using the ADSC-Quantum 315r CCD detectors (at 100 K). Data were processed with XDS software, and scaled using SCALA software from the CCP4 suite. Due to the weak diffraction of the ternary complex crystals, multiple crystals (∼10–15) were merged together, resulting in a multiplicity of almost 70. This allowed a gain in intensity, resolution as well as having better 2Fo-Fc and mFo-Fc density maps for the lipid antigen (). In a similar fashion, the CD1b–GMM data set represents a 360° sweep of data to improve the density of the lipid by increasing the multiplicity from a single crystal. The density for the acyl tails and the ester were clear; however the glucose moiety is solvent exposed and highly mobile. The glucose is most likely adopting a multitude of alternate conformations. Due to the high resolution and high multiplicity, we were able to build one conformation described here () that represents ∼20–30% occupation of the glucose moiety and was the only clear conformation that we could build. In turn the glucose is bulging out of the cleft, and is not stabilised by interaction with either the CD1b molecule itself or crystal packing, resulting in a mobile head group and weak density around it. The GEM42 TCR structure was determined by molecular replacement using PHASER program with the previously solved free GEM42 TCR as the search model for the TCR (Protein Data Bank accession number, 4G8F (ref. )), and we used previously solved CD1b structure as the search model (Protein Data Bank accession number, 1UQS (ref. )). Manual model building was conducted using the Coot software followed by maximum-likelihood refinement with Buster. The final models have been deposited to the Protein Data Bank under the accession code: 5L2J for CD1b–GMM (Ramachandran plot: 0% outlier) and 5L2K for GEM42 TCR–CD1b–GMM (Ramachandran plot: 0.9% outlier), and the final refinement statistics are summarized in . All molecular graphics representations were created using PyMol (DeLano WL. The PyMOL Molecular Graphics System. http://www.pymol.org 2002). The buried surface area is calculated with the AreaIMol program, the contacts generated by the Contact program, both form the CCP4 suite. The structures used to generate the comparison and figures are the MAIT-MR1-metabolite (PDB code: 4PJ7) (ref. ); ELS4-MHC-peptide (PDB code: 2NX5) (ref. ); BK6-CD1a-lipid (PDB code: 4X6C) (ref. ) and RL42-MHC-peptide (PDB code: 3SJV) (ref. ). […]

Pipeline specifications

Software tools XDS, CCP4, Coot, PyMOL
Organisms Mycobacterium, Homo sapiens
Diseases Tuberculosis
Chemicals Glucose