7QT1

Non-obligately L8S8-complex forming RubisCO derived from ancestral sequence reconstruction and rational engineering in L8S8 complex with substitution e170N

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, SITTING DROP	9.1	288	Purified enzyme (3.25 mg/mL) in 25 mM Tricine-NaOH, 75 mM NaCl, pH 8.0 was incubated at 3% (v/v) CO2 in air and 30 degrees C for 1 h in the presence of 0.182 mM CABP and 2.9 mM MgCl2. The enzyme was then mixed in a 1:1 ratio with 0.2 M Bis-Tris propane, 20 % (w/v) polyethylene glycol 4000, pH 9.1. Before flash freezing the crystals in liquid nitrogen PEG200 was added to the mother liquor as cryoprotectant to a final concentration of 33 % (w/v).

Crystal Properties
Matthews coefficient	Solvent content
2.17	43.23

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 108.942	α = 90
b = 106.604	β = 98.78
c = 191.968	γ = 90

Symmetry
Space Group	I 1 2 1

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 PILATUS3 2M		2021-12-02	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	ESRF BEAMLINE ID23-2	0.873128	ESRF	ID23-2

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Sym I (Observed)	Rrim I (All)	Rpim I (All)	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	2.1	29.29	99.9	0.218	0.239	0.096	7.1	6		126281

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	R-Sym I (Observed)	Rrim I (All)	Rpim I (All)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	2.1	2.21	100		0.71	0.71	0.777	0.311	1.1	6.1

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	6URA	2.1	29.29	1	126265	2000	99.9	0.1633	0.1629	0.1857	17.7926

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	17541
Nucleic Acid Atoms
Solvent Atoms	1601
Heterogen Atoms	88

Software

Software
Software Name	Purpose
SCALA	data scaling
PHENIX	refinement
PDB_EXTRACT	data extraction
XDS	data reduction
PHENIX	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.