8QMT

Succinic semialdehyde dehydrogenase from E. coli with Q262R substitution and bound NAD+, succinic semialdehyde

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	PETRA III, EMBL c/o DESY BEAMLINE P13 (MX1)
Synchrotron site	PETRA III, EMBL c/o DESY
Beamline	P13 (MX1)
Temperature [K]	100
Detector technology	PIXEL
Collection date	2023-06-20
Detector	DECTRIS EIGER X 16M
Wavelength(s)	0.9763
Spacegroup name	P 21 21 21
Unit cell lengths	91.710, 115.110, 179.380
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	29.550 - 1.800
R-factor	0.1805
Rwork	0.180
R-free	0.20540
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.006
RMSD bond angle	0.871
Data reduction software	XDS
Data scaling software	XSCALE
Phasing software	PHENIX (1.20.1_4487)
Refinement software	PHENIX (1.20.1_4487)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	29.550	29.550	1.840
High resolution limit [Å]	1.800	8.030	1.800
Rmerge	0.098	0.026	1.600
Rmeas	0.102	0.027	1.663
Number of reflections	174478	2136	12625
<I/σ(I)>	16.73
Completeness [%]	98.6
Redundancy	13.8
CC(1/2)	0.999	1.000	0.842

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7.5	295	14.8 mg/mL of SAD_Q262R in 20 mM HEPES-KOH, 50 mM KCl, pH 7.5 was complemented with 5 mM NAD+. The enzyme was then mixed in a 2:1 ratio with 250 mM Bis-Tris-Propane, pH 7.5 , 20 % (w/v) PEG4000. The final drop size was 3 uL. The drop was complemented with 6 mM succininc semialdehyde and 25 % (v/v) PEG400 before flash freezing the crystals in liquid nitrogen.