Computational protocol: A gatekeeper helix determines the substrate specificity of Sjögren–Larsson Syndrome enzyme fatty aldehyde dehydrogenase

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Protocol publication

[…] The FALDH protein was assayed for crystallization High Throughput Crystallization Laboratory of the EMBL Grenoble Outstation (https://embl.fr/htxlab) by the vapour diffusion method. Single three-dimensional crystals were obtained by mixing 100 nl of protein solution at a concentration of 15 mg ml−1 and 100 nl of crystallization solution (0.2 M Li2SO4, 0.1 M CHES pH9.5, 1.0 M K/Na tartrate). Crystals were transferred to a solution containing crystallization solution supplemented with 15% glycerol and cryo-cooled in liquid nitrogen. The FALDH crystals belonged to space group P 212121 and diffracted up to a resolution of 2.1 Å. X-ray diffraction experiments were performed at the beamline PROXIMA1 of the synchrotron SOLEIL and the MX beam lines of the European Synchrotron Radiation Facility (ESRF). Crystallographic data reduction and scaling was carried out with the software XDS. Initial phases were obtained by the molecular replacement method using Phaser and the atomic coordinates of the human ALDH3A1 protein (3SZA) as search model. Successive rounds of automatic refinement and manual building were carried out with RefMac5 (ref. ) and Coot. Illustrations were produced with Pymol (http://www.pymol.org) and MOLE. Multiple Sequence Alignment of FALDH and ALDH3A1 from different species were calculated with the Clustal Omega (http://www.ebi.ac.uk/) and the following sequences: CAG33703.1, NP_031463.2, NP_113919.2, AAI49985.1, NP_001128640.1, NP_114178.1, NP_001159985.1. [...] Differences in fold stability of wild-type and mutated FALDH was estimated using empirical FoldX force field version 3.0 allowing for quantitative estimation of differences in protein fold stability. After a relaxation of the structure in the FoldX force field using ‘repairpdb’, in silico mutations were calculated allowing neighbouring residues to rearrange. To test robustness of FoldX predictions, we performed five individual runs for each of the two subunits of FALDH, leading to 10 individual estimations per mutation. Water molecules resolved in the crystal structure were retained in calculations, the ionic strength of the surrounding solution was set to default 0.050 M. Temperature was set to 298 K, the pH to 7.0. […]

Pipeline specifications

Software tools XDS, REFMAC5, Coot, PyMOL, Clustal Omega, FoldX
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Diseases Skin Diseases, Sjogren-Larsson Syndrome
Chemicals Acetaldehyde