8BWG

HRas (1-166) Y64 phosphorylation

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	DIAMOND BEAMLINE I03
Synchrotron site	Diamond
Beamline	I03
Temperature [K]	100
Detector technology	PIXEL
Collection date	2021-01-29
Detector	DECTRIS EIGER2 XE 16M
Wavelength(s)	0.9763
Spacegroup name	H 3 2
Unit cell lengths	92.662, 92.662, 119.323
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	47.925 - 1.320
Rwork	0.135
R-free	0.17390
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.010
RMSD bond angle	1.607
Data reduction software	DIALS
Data scaling software	Aimless
Phasing software	MOLREP
Refinement software	REFMAC (5.8.0352)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	47.925	47.880	1.340
High resolution limit [Å]	1.320	7.230	1.320
Rmerge	0.041	0.022	1.340
Rmeas	0.043	0.023	1.535
Rpim	0.014	0.008	0.723
Number of reflections	45988	314	2073
<I/σ(I)>	28.6
Completeness [%]	99.2
Redundancy	17.6	17.4	7.8
CC(1/2)	1.000	0.999	0.577

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP		293	Using a commercial crystal screen (HAMPTON RESEARCH, HR2-130) yielded a hit for monophosphorylated HRas under sitting drop conditions (drop size 600 nL) with a 1:1 ratio of protein solution (phospho-HRas 0.4 mM, RasGAP 0.4 mM, Na-HEPES 20 mM pH = 8.0, MgCl2 5 mM, NaF 20 mM) and precipitant (Na-citrate 100 mM pH = 5.6, Li2SO4 1.0 M, CaCl2 200 mM). After three rounds of microseeding well-formed single crystals were obtained using 2.0 uL sitting drops and a 1:1 ratio of protein buffer (HRas 400 uM, RasGAP 400 uM, MgCl2 5 mM, Na-HEPES 20 mM pH = 8.0, NaF 20 mM) and precipitant (Na-Citrate 100 mM pH = 5.6, Li2SO4 800 mM, CaCl2 200 mM). These were harvested using cryoprotectant (80% precipitant, 20% glycerol (v/v)) and sent for data collection.