6YUA

CO-dehydrogenase coupled to the N-terminal domain of the Acetyl-CoA synthase from Clostridium autoethanogenum isolated after tryptic digestion.

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, SITTING DROP	7.6	291.15	Crystallization was performed in an anoxic chamber (N2/H2, 95:5 %) with anaerobic solutions. Crystals were obtained by initial screening at 291.15 K using the sitting drop method on a 96-well MRC 2 Crystallization Plates in polystyrene (SWISSCI). The crystallization reservoir contained 90 uL of mother liquor, crystallization drop contained a mixture of 0.6 uL protein and 0.6 uL precipitant. The protein concentration was 28.5 mg/mL in 25 mM Tris pH 7.6, 10% (v/v) glycerol and 2 mM dithiothreitol. The best diffracting crystal of CODH bound to the ACS N-terminal domain was obtained by initial screening using the Shotgun (SG1) screen from Molecular dimensions. The crystallization reservoir contained 200 mM Magnesium chloride hexahydrate and 20% (w/v) polyethylene glycol 3350. The crystal was cryoprotected in the same crystallization solution supplemented with 25% ethylene glycol.

Crystal Properties
Matthews coefficient	Solvent content
2.61	52.98

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 89.333	α = 90
b = 89.333	β = 90
c = 527.13	γ = 90

Symmetry
Space Group	P 41 21 2

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	100	PIXEL	DECTRIS PILATUS 2M-F		2019-12-16	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	SLS BEAMLINE X06DA	1.00000	SLS	X06DA

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge I (Observed)	Rrim I (All)	Rpim I (All)	CC (Half)	Net I Over Average Sigma (I)	Redundancy	Number Reflections (All)	Number Reflections (Observed)	Observed Criterion Sigma (F)	Observed Criterion Sigma (I)	B (Isotropic) From Wilson Plot
1	3.14	131.783	84.3	0.26	0.268	0.061	0.999	8.9	17.7		17564

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	Rrim I (All)	Rpim I (All)	CC (Half)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	3.14	3.576	81.2		1.492	1.524	0.309	0.748	2.5	23.6

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Cut-off Sigma (F)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (Observed)	R-Work	R-Free	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	THROUGHOUT	6YTT	3.16	39.95	1.35	17526	893	45.91	0.2252	0.2238	0.252	65.4363

Temperature Factor Modeling
Anisotropic B[1][1]	Anisotropic B[1][2]	Anisotropic B[1][3]	Anisotropic B[2][2]	Anisotropic B[2][3]	Anisotropic B[3][3]

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	13810
Nucleic Acid Atoms
Solvent Atoms
Heterogen Atoms	62

Software

Software
Software Name	Purpose
PHENIX	refinement
PDB_EXTRACT	data extraction
XDS	data reduction
STARANISO	data scaling
MOLREP	phasing

RCSB PDB Core Operations are funded by the U.S. National Science Foundation (DBI-2321666), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.