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.