6U9D
Saccharomyces cerevisiae acetohydroxyacid synthase
Experimental procedure
| Experimental method | SINGLE WAVELENGTH |
| Source type | SYNCHROTRON |
| Source details | AUSTRALIAN SYNCHROTRON BEAMLINE MX1 |
| Synchrotron site | Australian Synchrotron |
| Beamline | MX1 |
| Temperature [K] | 100 |
| Detector technology | CCD |
| Collection date | 2018-10-26 |
| Detector | ADSC QUANTUM 210r |
| Wavelength(s) | 0.9537 |
| Spacegroup name | C 1 2 1 |
| Unit cell lengths | 368.647, 230.307, 183.532 |
| Unit cell angles | 90.00, 94.57, 90.00 |
Refinement procedure
| Resolution | 49.110 - 3.194 |
| R-factor | 0.2053 |
| Rwork | 0.205 |
| R-free | 0.25220 |
| Structure solution method | MOLECULAR REPLACEMENT |
| Starting model (for MR) | BSM complex18 (PDB code 5FEM) and the RSU of Thermotoga maritima16 (PDB code 2FGC). |
| RMSD bond length | 0.002 |
| RMSD bond angle | 0.585 |
| Data reduction software | XDS |
| Data scaling software | XDS |
| Phasing software | PHENIX |
| Refinement software | PHENIX (1.9_1692) |
Data quality characteristics
| Overall | Inner shell | Outer shell | |
| Low resolution limit [Å] | 49.115 | 49.110 | 3.250 |
| High resolution limit [Å] | 3.190 | 17.490 | 3.190 |
| Rmerge | 0.141 | 0.034 | 0.773 |
| Rmeas | 0.151 | 0.037 | 0.829 |
| Rpim | 0.054 | 0.015 | 0.299 |
| Total number of observations | 10100 | 90467 | |
| Number of reflections | 250942 | 1511 | 12014 |
| <I/σ(I)> | 11 | 38.3 | 2 |
| Completeness [%] | 99.5 | 94.4 | 96.5 |
| Redundancy | 7.7 | 6.7 | 7.5 |
| CC(1/2) | 0.996 | 0.997 | 0.833 |
Crystallization Conditions
| crystal ID | method | pH | temperature | details |
| 1 | VAPOR DIFFUSION, HANGING DROP | 291 | The crystals of Saccharomyces cerevisiae AHAS complex were obtained by the hanging drop vapor diffusion method. The catalytic subunit (70.527 kDa, 70 mg/ml) and regulatory subunit (29.625 kDa, 19.8 mg/ml) freshly thawed were mixed together in a ratio of 1:2, giving a solution where the concentration of the RSU was ~ 30% in excess compared to the CSU. The cofactors, inhibitor, and DTT were added to the solution to a final concentration of 2 mM ThDP, 1 mM FAD, 10 mM MgCl2, 1 mM BSM and 5 mM DTT. The crystallization buffer consisted of 0.2 M sodium malonate pH 7 and 20% w/v PEG 3,350. The crystallization drops consisted of equal volumes (1 ul) of well solution and enzyme complex |
