8ESS

Myoglobin variant Mb-cIII complex

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	SSRL BEAMLINE BL9-2
Synchrotron site	SSRL
Beamline	BL9-2
Temperature [K]	100
Detector technology	PIXEL
Collection date	2020-07-14
Detector	DECTRIS PILATUS 6M
Wavelength(s)	0.9795
Spacegroup name	P 6
Unit cell lengths	90.701, 90.701, 45.451
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	39.340 - 1.400
R-factor	0.1531
Rwork	0.152
R-free	0.17810
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	6m8f
RMSD bond length	0.010
RMSD bond angle	1.022
Data reduction software	XDS
Data scaling software	Aimless
Phasing software	PHENIX
Refinement software	PHENIX (1.13_2998)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	39.340	1.420
High resolution limit [Å]	1.400	1.400
Rmerge	0.069	1.742
Rpim	0.016	0.398
Number of reflections	42175	2160
<I/σ(I)>	27.2	2
Completeness [%]	100.0
Redundancy	20.2
CC(1/2)	1.000	0.714

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION		296	Mb(H64G,V68A) at 2.5 mM in 20 mM Tris-HCl pH 8.4, 1 mM EDTA, was mixed with an equal volume of reservoir solution (200 mM Tris-HCl pH 8.9 and 2.4 M ammonium sulfate) to perform hanging-drop vapor diffusion crystallization. A crystal was transferred from the drop it was grown in to another 4 uL drop containing well solution with the addition of 10% glycerol. The drop was overlaid with 4 uL mineral oil. A micropipette tip was dipped in sodium dithionite powder and the tip was then slowly submerged into the drop to allow the dithionite crystals to be released. 1 uL of benzyl diazoketone at 500 mM (dissolved in isopropanol) was then added to the drop for a final concentration of 100 mM, then incubated for an additional 22.5 minutes. The crystal was then looped and flash-frozen in liquid nitrogen.