8Q77
STRUCTURE OF PROTEIN KINASE CK2 CATALYTIC SUBUNIT (ISOFORM CK2ALPHA'; CSNK2A2 GENE PRODUCT) IN COMPLEX WITH THE BISUBSTRATE INHIBITOR ARC-780
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | SYNCHROTRON |
Source details | ESRF BEAMLINE MASSIF-3 |
Synchrotron site | ESRF |
Beamline | MASSIF-3 |
Temperature [K] | 100 |
Detector technology | PIXEL |
Collection date | 2020-11-14 |
Detector | DECTRIS EIGER X 4M |
Wavelength(s) | 0.9677 |
Spacegroup name | P 1 |
Unit cell lengths | 46.603, 47.919, 51.031 |
Unit cell angles | 66.59, 89.67, 88.16 |
Refinement procedure
Resolution | 41.250 - 1.255 |
R-factor | 0.1202 |
Rwork | 0.120 |
R-free | 0.15700 |
Structure solution method | MOLECULAR REPLACEMENT |
RMSD bond length | 0.023 |
RMSD bond angle | 1.772 |
Data reduction software | XDS |
Data scaling software | Aimless |
Phasing software | PHASER |
Refinement software | PHENIX (1.20.1_4487) |
Data quality characteristics
Overall | Outer shell | |
Low resolution limit [Å] | 46.578 | 1.364 |
High resolution limit [Å] | 1.255 | 1.255 |
Rmerge | 0.052 | 0.559 |
Rmeas | 0.061 | 0.648 |
Rpim | 0.031 | 0.326 |
Number of reflections | 80300 | 4015 |
<I/σ(I)> | 12.2 | 1.6 |
Completeness [%] | 72.5 | |
Redundancy | 3.9 | |
CC(1/2) | 0.998 | 0.806 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, SITTING DROP | 293 | THE CK2ALPHA' SOLUTION AFTER PROTEIN PURIFICATION (5 MG/ML CK2ALPHA' IN 500 MM NACL, 25 MM TRIS/HCl, PH 8.5) WAS MIXED IN 1:10 VOLUME RATIO WITH 10 MM SOLUTION OF THE INHIBITOR MB002 IN DMSO. THIS SOLUTION WAS MIXED IN 2:1 RATIO WITH RESERVOIR SOLUTION [900 MM LICL, 28 % (W/V) PEG 6000, 250 MM TRIS/HCL, PH 8.5] IN SITTING DROP PLATES (VAPOUR DIFFUSION). THE CRYSTAL GROWTH WAS INDUCED BY MICROSEEDING. THE CRYSTALS WERE OPTIMIZED BY MACROSEEDING AND PURGED TWICE WITH RESERVOIR SOLUTION. THEN, ARC-780 WAS ADDED TO AN INITIAL CONCENTRATION OF 1 MM BY ADDING 1 MIKROLITER OF A 10 MM ARC-780 SOLUTION IN DMSO IN A 1:10 RATIO TO THE DROPLET. AFTER EXTENSIVE INTIAL SOAKING, THE SALT CONCENTRATION WAS SUCCESSIVELY LOWERED OVER A PERIOD OF 8 MONTHS WHILE IN PARALLEL THE CONCENTRATIONS OF DMSO AND PEG 6000 WERE INCREASED. THIS WAS DONE BY GRADUALLY REPLACING THE RESERVOIR AND THE DROPLET SOLUTION UNTIL ANY NACL WAS REMOVED, THE LICL CONCENTRATION WAS REDUCED TO 50 MM, THE PEG 6000 CONCENTRATION WAS INCREASED TO SATURATION AND A DMSO CONTENT OF 30% WAS REACHED. MEANWHILE, THE ARC-780 LIGAND WAS INCLUDED IN ANY DESALTING STEP REACHING SATURATION AFTER THE FINAL DILUTION STEP. ALL STEPS WERE PERFORMED AT A TEMPERATURE OF 293 K. |