8OOO

Glutamine synthetase from Methanothermococcus thermolithotrophicus in complex with 2-oxoglutarate and MgATP at 2.15 A resolution

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	2020-08-27
Detector	DECTRIS PILATUS 2M-F
Wavelength(s)	1.30511
Spacegroup name	P 1
Unit cell lengths	112.337, 131.773, 131.507
Unit cell angles	60.04, 87.72, 67.34

Refinement procedure

Resolution	42.880 - 2.149
R-factor	0.1832
Rwork	0.182
R-free	0.21400
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.009
RMSD bond angle	0.930
Data reduction software	autoPROC
Data scaling software	STARANISO
Phasing software	PHASER
Refinement software	BUSTER (2.10.4 (21-NOV-2022))

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	110.034	2.297
High resolution limit [Å]	2.148	2.148
Rmerge	0.110	0.950
Rmeas	0.130	1.122
Rpim	0.068	0.590
Number of reflections	248419	12420
<I/σ(I)>	9.3	1.3
Completeness [%]	92.2	58.8
Redundancy	3.6	3.4
CC(1/2)	0.995	0.569

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7.6	293.15	The enzyme was crystallized at 3.7 mg/ml with a final concentration of 2 mM sodium 2-oxoglutarate, 2 mM ATP and 2 mM MgCl in 25 mM Tris/HCl pH 7.6, 10% v/v glycerol, 150 mM NaCl, 2 mM dithiothreitol. The protein was crystallized fresh without any freezing step, and crystallization was performed through the sitting drop method on a 96-Well MRC 2-Drop Crystallization Plates in polystyrene (SWISSCI, United Kingdom) under anaerobic conditions (N2:H2, gas ratio of 97:3). The crystallization reservoir contained 90 ul of mother liquor (20 % w/v polyethylene glycol 3,350 and 200 mM sodium fluoride). The crystallization drop contained 0.6 ul protein with ligands and 0.6 ul precipitant. Crystals were soaked in the mother liquor supplemented with 20 % v/v glycerol prior to freezing in liquid nitrogen.