Crystallization experiments and structure determination were conducted in the Collaborative Crystallography Core in the Department of Biochemistry at the University of Wisconsin. All crystallization screens and optimizations were conducted at 293K in MRC SD-2 crystallization plates, set with a STP Labtech Mosquito crystallization robot. Hampton IndexHT and Molecular Dimensions JCSG+ were used as general screens. Crystals were cooled by direct immersion in liquid nitrogen after cryopreservation and harvest using MiTeGen MicroMounts. X-ray experiments were conducted at the Advanced Photon Source, Argonne National Lab, GMCA@APS beamline 23ID-D. Diffraction data were collected on a Pilatus 3-6M detector and reduced using XDS (79 (link)) and XSCALE (80 (link)). Structure solution and refinement used the Phenix suite of crystallography programs (81 ). Iterative rounds of map fitting in Coot (82 (link)) and phenix.refine (83 (link)) were used to improve the atomic models. MOLPROBITY (84 (link)) was used to validate the structures. Data collection and refinement statistics for the structures can be found in Table S3 .
The best crystals of SeMet Aim18p-Nd70-R123A grew from 26% PEG 3350, 0.2 M Li2SO4, 0.1 M Na-Hepes buffer, pH 7.5. Crystals were cryopreserved by supplementing the PEG concentration to 30%. Diffraction data extending to 2.2 Å was collected on 2018-02-07 at energies near the SeK-edge (peak = 0.97937 Å, edge = 0.97961 Å, and high remote at 0.96437 Å). Phenix.hyss (85 (link)) located the expected 5 Se sites from one copy of the protein per asymmetric unit in space group P3221. The structure was phased and traced using Phenix.autosol (86 (link)) with a phasing figure of merit = 0.47 and map skew = 0.6. The initial chain trace was continuous from residue 88 to the C-terminus. The final model starts at residue 83 and includes five sulfate ions per chain.
The best crystals of Aim46p-FL-WT were grown using microseeds obtained from a similar condition. The seeds were stabilized in 30% PEG 2000, 0.1 M MES pH 6.5. The droplet was composed of 20 nl microseed suspension, 180 nl Aim46p-Nd62-WT, and 300 nl PEG 2000, 0.1 M MES, pH 6.5. Crystals were cryoprotected with 30% PEG 2000. Data were collected on 2018-12-08 at 1.0332 Å. Data extended to 2.0 Å and belonged to space group P21. One copy of the protein per asymmetric unit was by molecular replacement using Phaser starting from an appropriately pruned Aim18p-Nd70-R123A model (log-likelihood gain 307, translation function z-score 11.7). The final model is continuous from residue 70 to the C-terminus and includes a ligand modeled as α-ketoglutarate.
The best crystals of SeMet Aim18p-Nd70-R123A grew from 26% PEG 3350, 0.2 M Li2SO4, 0.1 M Na-Hepes buffer, pH 7.5. Crystals were cryopreserved by supplementing the PEG concentration to 30%. Diffraction data extending to 2.2 Å was collected on 2018-02-07 at energies near the SeK-edge (peak = 0.97937 Å, edge = 0.97961 Å, and high remote at 0.96437 Å). Phenix.hyss (85 (link)) located the expected 5 Se sites from one copy of the protein per asymmetric unit in space group P3221. The structure was phased and traced using Phenix.autosol (86 (link)) with a phasing figure of merit = 0.47 and map skew = 0.6. The initial chain trace was continuous from residue 88 to the C-terminus. The final model starts at residue 83 and includes five sulfate ions per chain.
The best crystals of Aim46p-FL-WT were grown using microseeds obtained from a similar condition. The seeds were stabilized in 30% PEG 2000, 0.1 M MES pH 6.5. The droplet was composed of 20 nl microseed suspension, 180 nl Aim46p-Nd62-WT, and 300 nl PEG 2000, 0.1 M MES, pH 6.5. Crystals were cryoprotected with 30% PEG 2000. Data were collected on 2018-12-08 at 1.0332 Å. Data extended to 2.0 Å and belonged to space group P21. One copy of the protein per asymmetric unit was by molecular replacement using Phaser starting from an appropriately pruned Aim18p-Nd70-R123A model (log-likelihood gain 307, translation function z-score 11.7). The final model is continuous from residue 70 to the C-terminus and includes a ligand modeled as α-ketoglutarate.
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