Computational protocol: Improvements in the order, isotropy and electron density of glypican-1 crystals by controlled dehydration

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Protocol publication

[…] Saturated ammonium sulfate and sodium chloride salt solutions were used to calibrate the HC1 machine installed online at station I911-3 of the MAX IV Laboratory, Lund, Sweden (Ursby et al., 2013) as described previously (Sanchez-Weatherby et al., 2009). The approximate RH of the Gpc-1 cryosolution (13% PEG 6000, 0.2 M CaCl2, 0.1 M Tris pH 8.0, 15% ethylene glycol) was found by running the double-gradient script to adjust the RH from 99 to 90% RH and back again while monitoring the size of the drop using the HC1 software. The RH of the humid air was modified until it was in equilibrium with the drop. The drop remained the same size between 95 and 96% RH and this was thus used as the initial RH (RHi) for all further experiments.Gpc-1 crystals were mounted on mesh LithoLoops (Molecular Dimensions, Newmarket, England) of sizes 0.2 and 0.3 mm, briefly soaked in the cryosolution and finally mounted in the HC1 air stream at RHi = 95% for dehydration. Excess liquid was removed from the opposite side of the mesh loop to the crystal using a paper wick. An initial diffraction image was collected at room temperature to judge the crystal quality. The smallest available beam diameter of 30 µm combined with short X-ray exposures (about 5–10 s per image) was used to expose the crystal minimally and limit radiation damage yet still permit successful indexing. Lattice parameters and relative diffraction resolution were monitored using iMosflm (Battye et al., 2011) to assess lattice changes. Six to ten images were typically collected at room temperature from each Gpc-1 crystal at different relative humidities, translating the crystal by more than 50 µm between exposures.The variations in the dehydration protocols for each experiment are described in §3. Briefly, a set of parameters was tested, including the final RH (RHf; from 85 to 95%), the dehydration rate (per 0.1% RH; 10–90 s) and lastly the total incubation time T inc of the crystal in the humid air stream, including all dehydration and equilibration, before crystal cooling. After dehydration (without exposure to X-rays), the crystal was unmounted into an empty vial containing liquid N2 using the CATS sample changer (IRELEC, Saint-Martin-d’Hères, France) and stored in liquid N2 for subsequent diffraction testing. Once a sufficient number of crystals had been harvested, the Cryostream (Oxford Cryosystems, Oxford, England) was remounted and diffraction data were collected from each crystal at 100 K. [...] X-ray diffraction data were collected from cooled dehydrated crystals at 100 K on station I911-3. Diffraction images were indexed, integrated and scaled using XDS (Kabsch, 2010) and were further processed using programs from the CCP4 (Winn et al., 2011) and PHENIX (Adams et al., 2010) packages. Analysis of X-ray data sets was performed using phenix.xtriage and SFCHECK (Vaguine et al., 1999). The structures were solved using AutoMR in PHENIX with Phaser (McCoy et al., 2007) against a dimer consisting of chains C and D of PDB entry 4acr as a starting model. The initial models were completed by manual building in Coot (Emsley et al., 2010) followed by rounds of refinement using REFMAC5 (Murshudov et al., 2011) and, in the final stages, phenix.refine (Afonine et al., 2012). The models were validated using MolProbity (Chen et al., 2010). Graphical representations were generated using Coot and the PyMOL Molecular Graphics System (v.1.5; Schrödinger, New York, USA). Structural alignments were created in PyMOL via an initial sequence alignment. Crystal packing and total interface area were evaluated by PISA analysis (Krissinel & Henrick, 2007). The coordinates and the diffraction data of the glypican-1 structure from crystals dehydrated to 86% using the optimal protocol have been deposited in the Protein Data Bank with accession code 4bwe. […]

Pipeline specifications

Software tools iMosflm, XDS, CCP4, PHENIX, Coot, REFMAC5, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens