6OW4

Structure of the NADH-bound form of 20beta-Hydroxysteroid Dehydrogenase from Bifidobacterium adolescentis strain L2-32

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 21-ID-D
Synchrotron site	APS
Beamline	21-ID-D
Temperature [K]	100
Detector technology	PIXEL
Collection date	2018-03-17
Detector	DECTRIS EIGER X 9M
Wavelength(s)	1.07
Spacegroup name	C 1 2 1
Unit cell lengths	165.078, 134.710, 96.396
Unit cell angles	90.00, 100.15, 90.00

Refinement procedure

Resolution	103.710 - 1.990
R-factor	0.19
Rwork	0.187
R-free	0.23840
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	6m9u
RMSD bond length	0.003
RMSD bond angle	1.188
Data reduction software	xia2
Data scaling software	autoPROC
Phasing software	PHENIX
Refinement software	REFMAC (5.8.0238)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	103.710	2.045
High resolution limit [Å]	1.990	1.990
Number of reflections	73226	3662
<I/σ(I)>	6.3
Completeness [%]	54.3
Redundancy	4.8
CC(1/2)	0.995

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP		296	Mutant S181A 20beta-HSDH purified recombinant protein was incubated with 2.5 mM NADH and 0.25mM or 0.5 mM cortisol for 2 hours at 4C. Crystals were grown in condition 86 of the Hampton PEG/Ion screen containing 0.05 M Citric acid, 0.05 M BIS-TRIS propane / pH 5.0, and 16% w/v Polyethylene glycol 3,350. The condition was then optimized in hanging-drop format using 18-20% w/v Polyethylene glycol 3,350