8CNS

The Hybrid Cluster Protein from the thermophilic methanogen Methanothermococcus thermolithotrophicus in a mixed redox state after soaking with hydroxylamine, at 1.36-A resolution.

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	PETRA III, DESY BEAMLINE P11
Synchrotron site	PETRA III, DESY
Beamline	P11
Temperature [K]	100
Detector technology	PIXEL
Collection date	2020-06-07
Detector	DECTRIS PILATUS 6M-F
Wavelength(s)	1.000400
Spacegroup name	P 21 21 2
Unit cell lengths	97.773, 102.186, 58.323
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	51.090 - 1.360
R-factor	0.1309
Rwork	0.129
R-free	0.16610
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.012
RMSD bond angle	1.352
Data reduction software	autoPROC
Data scaling software	autoPROC
Phasing software	PHASER
Refinement software	PHENIX (1.20.1-4487)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	70.645	1.476
High resolution limit [Å]	1.356	1.356
Rmerge	0.110	1.061
Rmeas	0.118	1.165
Rpim	0.043	0.472
Number of reflections	89372	4469
<I/σ(I)>	11.5	1.6
Completeness [%]	94.9	67.9
Redundancy	7.1	5.9
CC(1/2)	0.999	0.611

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7.6	293.15	Crystallisation was performed anaerobically by initial screening at 20 degree Celsius using the sitting drop method on 96-Well MRC 2-Drop polystyrene Crystallisation Plates (SWISSCI) in a Coy tent containing a N2/H2 (97:3%) atmosphere. The reservoir chamber was filled with 90 ul of crystallisation condition, and the crystallisation drop was formed by spotting 0.55 ul protein with 0.55 ul of 20% (w/v) PEG 3,350 and 200 mM Magnesium formate. The protein was crystallised at 9.9 mg/ml in 25 mM Tris/HCl pH 7.6, 2 mM dithiothreitol, 10% (v/v) glycerol. Densities in the electron density map suggest a contamination of another crystallisation condition spatially close and containing 30% (v/v) 2-Methyl-2,4-pentanediol, 20 mM Calcium chloride and 100 mM Sodium acetate, pH 4.6. The crystal was soaked for 5.7 min in a solution of 100 mM hydroxylamine/HCl in the crystallisation condition and then soaked in the crystallisation solution supplemented with 20% v/v ethylene glycol for a few seconds before freezing in liquid nitrogen.