7B2C

Crystal structure of the ethyl-coenzyme M reductase from Candidatus Ethanoperedens thermophilum gassed with xenon

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	2019-09-29
Detector	DECTRIS PILATUS 2M-F
Wavelength(s)	1.54981
Spacegroup name	P 1 21 1
Unit cell lengths	83.791, 147.186, 113.376
Unit cell angles	90.00, 107.20, 90.00

Refinement procedure

Resolution	35.900 - 1.800
R-factor	0.176
Rwork	0.174
R-free	0.20100
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	7b1s
RMSD bond length	0.008
RMSD bond angle	0.990
Data reduction software	XDS
Data scaling software	STARANISO
Phasing software	PHENIX
Refinement software	BUSTER (2.10.3 (19-MAR-2020))

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	60.870	1.840
High resolution limit [Å]	1.800	1.800
Rmerge	0.105	0.652
Rpim	0.046	0.331
Number of reflections	232772	11640
<I/σ(I)>	11.7	2
Completeness [%]	98.1	94.6
Redundancy	6	4.5
CC(1/2)	0.997	0.739

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	8.5	293.15	Crystals were obtained by initial screening at 20 degree Celsius using the sitting drop method on a 96-Well MRC 2-Drop Crystallization Plates in polystyrene (SWISSCI). The crystallization reservoir contained 90 ul of mother liquor, crystallization drop contained a mixture of 0.6 ul protein at 16.22 mg.ml-1 and 0.6 ul precipitant. The crystal was obtained by initial screening using the JBScreen Pentaerythritol screen from Jena Bioscience in a Coy tent under an N2/H2 atmosphere (95:5 %). The crystallization reservoir contained 45 % (w/v) Pentaerythritol Propoxylate (5/4 PO/OH), 100 mM Tris pH 8.5 and 400 mM potassium chloride. Crystal was harvested using a MiTeGen MicroRT loop. In order to protect the crystal during xenon pressurisation, a part of the plastic capillary, usually used for room temperature diffraction experiments, was placed around it. The loop was then mounted in an Oxford Xcell xenon chamber. Xenon pressure was gradually increased using a manual pump to reach 25 bars. After 7 min, pressure was slowly released and the crystal immediately plunged in liquid nitrogen.