8BTS

Nitrogenase MoFe protein from A. vinelandii alpha double mutant C45A/L158C

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	SLS BEAMLINE X06DA
Synchrotron site	SLS
Beamline	X06DA
Temperature [K]	100
Detector technology	PIXEL
Collection date	2021-11-27
Detector	DECTRIS PILATUS 2M-F
Wavelength(s)	1.00003
Spacegroup name	P 1 21 1
Unit cell lengths	148.392, 73.444, 211.253
Unit cell angles	90.00, 104.76, 90.00

Refinement procedure

Resolution	95.500 - 3.030
R-factor	0.2046
Rwork	0.204
R-free	0.22270
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.004
RMSD bond angle	0.664
Data reduction software	autoPROC (1.0.5)
Data scaling software	autoPROC (1.0.5)
Phasing software	PHASER
Refinement software	PHENIX (1.20.1-4487)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	143.493	3.486
High resolution limit [Å]	3.029	3.029
Rmerge	0.297	0.514
Rmeas	0.346	0.615
Rpim	0.175	0.334
Number of reflections	39936	1998
<I/σ(I)>	3.4	1.6
Completeness [%]	86.6	69.4
Redundancy	3.7	3.3
CC(1/2)	0.957	0.806

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	5.5	293.15	The purified enzyme in 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 2 mM dithionite was crystallized at 8 mg.ml-1. Crystallization was performed anaerobically in a tent containing 100 % N2 atmosphere. Crystals were obtained by initial screening at 20 degree Celsius on 96-Well MRC 2-drop crystallization plates in polystyrene (SWISSCI) containing 90 ul of crystallization solution in the reservoir. The protein sample (0.5 ul) was mixed with 0.5 ul reservoir solution. The reservoir solution contained 25 % w/v Polyethylene glycol 3,350, 100 mM BIS-TRIS at pH 5.5, and 200 mM Lithium sulfate. The plate was then incubated in a Coy tent (N2/H2, 97:3) at 20 degree Celsius. Crystals appeared after a few weeks and were soaked in the crystallization solution supplemented with 15 % v/v glycerol as a cryo-protectant before being frozen in liquid nitrogen.