Computational protocol: Structural Basis of Substrate Recognition by AldehydeDehydrogenase 7A1

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Protocol publication

[…] X-ray diffraction data for C2 apo ALDH7A1 crystals were collected on Northeastern Collaborative Access Team beamline 24-ID-E at the Advanced Photon Source using a Q315r detector. The 2.4 Å resolution data set used for refinement consisted of 180 frames with an oscillation width of 1° per image, a detector distance of 310 mm, and an exposure time of 1 s per image.X-ray diffraction data from monoclinic and tetragonal crystals of the ALDH7A1–NAD+ complex were collected on beamline 4.2.2 at the Advanced Light Source using a Taurus-1 CMOS detector in shutterless mode. Each data set consisted of 1800 frames covering a total rotation range of 180° with the detector distance set to 240 mm. The total exposure times were 540 s for the C2 data set and 180 s for the tetragonal data set.X-ray diffraction data for crystals of the ALDH7A1–AA complex and tetragonal apo ALDH7A1 were collected on Structural Biology Center beamline 19-ID-C at the Advanced Photon Source using a Q315r detector. The 1.76 Å resolution data set used for refinement of the ALDH7A1–AA complex consisted of 720 frames with an oscillation width of 0.25° per image, a detector distance of 230 mm, and an exposure time of 1 s per image. The 1.90 Å resolution data set used for refinement of tetragonal apo ALDH7A1 consisted of 360 frames with an oscillation width of 0.5° per image, a detector distance of 260 mm, and an exposure time of 1 s per image.The data sets were processed with XDS and AIMLESS via CCP4i. PHENIX and COOT were used for refinement and model building, respectively. Crystallographic structure refinements against the C2 data sets were initiated from the coordinates of truncated ALDH7A1 [Protein Data Bank (PDB) entry 2J6L]. Initial phases for the tetragonal data sets were determined using molecular replacement as implemented in PHASER with a search model derived from a dimer of human ALDH7A1 (PDB entry 2J6L). MolProbity was used for structure validation. Data collection and refinement statistics are listed in and . [...] SAXS experiments were performed at beamline 12.3.1 of the Advanced Light Source via the mail-in program., Prior to data collection, an ALDH7A1 sample was passed through a Superdex 200 size exclusion column. The column buffer consisted of 50 mM Tris, 5% glycerol, 0.5 mM THP, and 50 mM NaCl (pH 7.8). Scattering intensities were measured at three nominal protein concentrations using exposure times of 0.5, 1.0, 3.0, and 6.0 s. Scattering curves collected from the protein samples were corrected for background scattering using intensity data collected from the Superdex 200 column effluent. Composite scattering curves for each protein concentration were generated with PRIMUS by scaling and merging the background-corrected high-q region from the 3 s exposure with the low-q region from the 0.5 or 1.0 s exposure. PRIMUS was also used for Guinier analysis. Shape reconstruction calculations were performed with the sastbx.shapeup module of the Small Angle Scattering ToolBox server. The shape reconstruction calculations employed the PISA database of shapes. The FoXS server was used to calculate SAXS profiles from atomic coordinates. […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, Coot, MolProbity, FoXS
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens
Diseases Epilepsy, Hyperlysinemias
Chemicals Acetaldehyde, NAD, Pyridoxine, Glutamic Acid