New insight into the dynamic properties and the active site architecture of H-Ras p21 revealed by X-ray crystallography at very high resolution

Table 1 Crystallization parameters for crystals C1 - C7.

crystal	nucleotide	c(protein) (mg/ml)	reservoir solution	cryo solution	reservoir volume	drop size	seeding technique	crystal age
C1	GppNHp	12.64	64 mM TRIS pH 7.6 20 mM MgCl₂ 10 mM DTT 0.1 mM NaN₃ 26% PEG 400	directly frozen	450 μl	2 μl	-	2 days
C2	GDP	17.27	64 mM TRIS pH 7.6 20 mM MgCl₂ 10 mM DTT 0.1 mM NaN₃ 35% PEG 400	directly frozen	500 μl	4 μl	-	6 months
C3	S-caged GTP	15.40	0.2 M Mg acetate 0.1 M HEPES pH 7.4 17% PEG 8000	XC-17/20 ^a	1 ml	20 μl	streak-seeding	3 days
C4	R-caged GTP	13.35	0.2 mM Mg acetate 0.1 M HEPES pH 7.2 16% PEG 8000	~3 min in XC-16/10 ^b, then transferred to XC-17/20 ^a	500 μl	4 μl	streak-seeding	3 days
C5	R-caged GTP→GTP	13.35	0.2 M Mg acetate 0.1 M HEPES pH 7.2 16% PEG 8000	XC-17/20 ^a	1 ml	4 μl	streak-seeding	3 days + ~4 min after photolysis
C6	R-caged GTP→GDP	13.35	0.2 M Mg acetate 0.1 M HEPES pH 7.2 16% PEG 8000	XC-17/20 ^a	1 ml	4 μl	streak-seeding	3 days + 68 hours after photolysis
C7	R-caged GTP→GTP	13.35	0.2 M Mg acetate 0.1 M HEPES pH 7.2 16% PEG 8000	XC-17/20 ^a	1 ml	4 μl	streak-seeding	3 days + ~3 min after photolysis

^a The composition of XC-17/20 cryo solution is 100 mM HEPES pH 7.2, 64 mM TRIS pH 7.6, 20 mM MgCl₂, 200 mM Mg-acetate, 0,1 mM NaN₃, 17% PEG 8000 (freshly prepared), 20% glycerol.
^b The composition of XC-16/10 cryo solution is 100 mM HEPES pH 7.2, 64 mM TRIS pH 7.6, 20 mM MgCl₂, 200 mM Mg-acetate, 0,1 mM NaN₃, 16% PEG 8000 (freshly prepared), 10% glycerol.

ISSN: 1472-6807