Experimental procedure
| Experimental method | SINGLE WAVELENGTH |
| Source type | SYNCHROTRON |
| Source details | PETRA III, DESY BEAMLINE P11 |
| Synchrotron site | PETRA III, DESY |
| Beamline | P11 |
| Temperature [K] | 100 |
| Detector technology | PIXEL |
| Collection date | 2020-04-13 |
| Detector | DECTRIS PILATUS 6M |
| Wavelength(s) | 1.0332 |
| Spacegroup name | C 1 2 1 |
| Unit cell lengths | 113.357, 53.113, 44.891 |
| Unit cell angles | 90.00, 102.98, 90.00 |
Refinement procedure
| Resolution | 23.930 - 1.800 |
| R-factor | 0.1849 |
| Rwork | 0.184 |
| R-free | 0.20670 |
| Structure solution method | MOLECULAR REPLACEMENT |
| Starting model (for MR) | 7ar6 |
| RMSD bond length | 0.010 |
| RMSD bond angle | 0.753 |
| Data reduction software | XDS |
| Data scaling software | XDS |
| Phasing software | PHASER |
| Refinement software | PHENIX (1.18_3845) |
Data quality characteristics
| Overall | Outer shell | |
| Low resolution limit [Å] | 47.870 | 1.830 |
| High resolution limit [Å] | 1.800 | 1.800 |
| Rmerge | 0.107 | 2.187 |
| Rmeas | 0.123 | 2.527 |
| Rpim | 0.060 | 1.245 |
| Number of reflections | 24189 | 1359 |
| <I/σ(I)> | 8.8 | 0.6 |
| Completeness [%] | 99.4 | 94.2 |
| Redundancy | 4 | 3.9 |
| CC(1/2) | 0.997 | 0.261 |
Crystallization Conditions
| crystal ID | method | pH | temperature | details |
| 1 | COUNTER-DIFFUSION | 291 | Co-crystallization with the compounds was achieved mixing 0.23 uL of protein solution (6.25 mg/mL) in 20 mM HEPES buffer (pH 7.8) containing 1 mM DTT/TCEP (respectively), 1 mM EDTA, and 150 mM NaCl with 0.22 uL of reservoir solution consisting of 100 mM MIB, pH 7.5, containing 25% w/w PEG 1500 and 5% (v/v) DMSO, and 0.05 uL of a micro-seed crystal suspension. This growth solution was equilibrated by sitting drop vapor diffusion against 40 uL reservoir solution. Prior to crystallization 125 nL droplets of 10 mM compound solutions from the two libraries in DMSO were applied to the wells of SwissCI 96-well plates (2-well or 3-well low profile, respectively) and subsequently dried in vacuum. Taking the crystallization drop volume into account this resulted in a final compound concentration of 2.5 mM and a molar ratio of 13.6 of compound to protein. To obtain well-diffracting crystals in a reproducible way micro-seeding was applied for crystal growth. Crystals appeared within a few hours and reached their final size (200x100x10 um3) after 2 - 3 days. Crystals were manually harvested and flash-frozen in liquid nitrogen for subsequent X-ray diffraction data collection. |
