Computational protocol: Structural insights into binding of STAC proteins to voltage-gated calcium channels

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Protocol publication

[…] The STAC3 SH3-2 domain was crystallized by using sitting-drop or hanging-drop vapor diffusion at room temperature by mixing equal volumes of protein (16 mg/mL) and well solution, containing 0.1 M Bis⋅Tris (pH 6.5), 22.5% (wt/vol) PEG 3350, and 0.2 M ammonium acetate. Crystals were soaked in a mixture of mother liquor and 30% ethylene glycol and flash-frozen. The tandem SH3 domain construct of STAC1 was crystallized by sitting-drop vapor diffusion at room temperature and by mixing equal volumes of protein (10 mg/mL) and well solution, which contained 0.1 M Na–Hepes (pH 7.5) and 25% (wt/vol) PEG 1000. Crystals were transferred to a cryosolution containing the mother liquor and 35% (vol/wt) glycerol and flash-frozen. The wild-type STAC2 tandem SH3 domain construct was crystallized in a 350-µL dialysis button against 150 mM KCl, 10 mM Hepes (pH 7.4), and 2 mM TCEP at 4 °C. Its complex with the synthetic minimal CaV1.1 peptide (obtained from LifeTein) was crystallized in a 1:2 ratio of STAC2:peptide by sitting-drop or hanging-drop vapor diffusion at 4 °C by mixing an equal amount of protein and well solution, which contained 2.24 M (NH4)2SO4 and 0.1 M sodium acetate (pH 5.5). Before being flash-frozen in liquid nitrogen, the apo and complex STAC2 crystals were transferred to a drop containing mother liquor and 35 and 30% (vol/vol) glycerol, respectively. The STAC2 Q347I mutant was crystallized by hanging-drop vapor diffusion at 4 °C upon mixing equal volumes of protein (20 mg/mL) and well solution [0.1 M Bis⋅Tris, pH 6.4, 0.2 M lithium sulfate, and 35% (wt/vol) PEG 3350]. The Q347I mutant was cryoprotected with 25% PEG 200 and mother liquor. Diffraction data were collected at the Stanford Synchrotron Radiation Lightsource beamline BL9-2, the Advanced Photon Source beamline 23-ID-D, the Canadian Light Source beamline 08ID-1, and our homesource (Micromax 007 HF, Mar345 detector).Data sets were processed by using XDS () and xia2. Initial phases were collected by iodide single-wavelength anomalous diffraction for the second SH3 domain of STAC3. Crystals of the second SH3 domain of STAC3 were soaked in 1 M NaI for 1–2 min before being frozen. Phases were determined via PHENIX (); ARP/wARP was used to build an initial model of the structure of this individual domain; and the subsequent models were refined with PHENIX using high-resolution native datasets at 1.3 Å. The structure of apo-STAC2 (tandem SH3 domains) was solved by molecular replacement via Phaser (), using the SH3-2 domain of STAC3 and an SH3 NMR structure (PDB ID code 2DL4) as search models. Structures of STAC2 Q347I, apo-STAC1, and the complexed STAC2 tandem SH3 domains were solved by molecular replacement using the structure of apo-STAC2 as a search model. All models were completed with iterative cycles of manual model building in Coot () and refinement with Refmac5 (). All structure files are available in the PDB with accession codes 6B25, 6B26, 6B27, 6B28, and 6B29. […]

Pipeline specifications

Software tools XDS, PHENIX, ARP/wARP, Coot, REFMAC5
Applications Small-angle scattering, Protein structure analysis
Organisms Canine mastadenovirus A
Diseases Muscular Diseases
Chemicals Calcium, Ryanodine