2LF0

Solution structure of sf3636, a two-domain unknown function protein from Shigella flexneri 2a, determined by joint refinement of NMR, residual dipolar couplings and small-angle X-ray scattering, NESG target SfR339/OCSP target sf3636

SOLUTION NMR - SOLUTION SCATTERING

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	3D HNCO	0.7 mM [U-100% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3-6, 10 mM benzamidine, 1 x inhibitor cocktail	90% H2O/10% D2O	300	7	ambient	298
2	3D CBCA(CO)NH	0.7 mM [U-100% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3-6, 10 mM benzamidine, 1 x inhibitor cocktail	90% H2O/10% D2O	300	7	ambient	298
3	3D HBHA(CO)NH	0.7 mM [U-100% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3-6, 10 mM benzamidine, 1 x inhibitor cocktail	90% H2O/10% D2O	300	7	ambient	298
4	3D HNCA	0.7 mM [U-100% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3-6, 10 mM benzamidine, 1 x inhibitor cocktail	90% H2O/10% D2O	300	7	ambient	298
5	3D HCCH-TOCSY	0.7 mM [U-100% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3-6, 10 mM benzamidine, 1 x inhibitor cocktail	90% H2O/10% D2O	300	7	ambient	298
6	3D CCH-TOCSY	0.7 mM [U-100% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3-6, 10 mM benzamidine, 1 x inhibitor cocktail	90% H2O/10% D2O	300	7	ambient	298
7	3D 1H-15N NOESY	0.7 mM [U-100% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3-6, 10 mM benzamidine, 1 x inhibitor cocktail	90% H2O/10% D2O	300	7	ambient	298
8	3D 1H-13C NOESY	0.7 mM [U-100% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3-6, 10 mM benzamidine, 1 x inhibitor cocktail	90% H2O/10% D2O	300	7	ambient	298
9	3D 1H-13C NOESY	0.5 mM [U-100% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 x inhibitor cocktail	100% D2O	300	7	ambient	298
10	3D 1H-13C NOESY aromatic	0.7 mM [U-100% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3-6, 10 mM benzamidine, 1 x inhibitor cocktail	90% H2O/10% D2O	300	7	ambient	298
11	2D 1H-13C HSQC	0.5 mM [U-7% 13C; U-100% 15N] sf3636, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 uM zinc sulphate, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 x inhibitor cocktail	90% H2O/10% D2O	300	7	ambient	298

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	AVANCE	800
2	Bruker	AVANCE	600

NMR Refinement
Method	Details	Software
simulated annealing	The solution structures were generated using CYANA 3.0 with automated noesy assignments and RDC restraints and further refined by incorporating RDC restraints and SAXS data in restrained molecular dynamics simulation with the program CNS. Although the current models fit well with both RDCs and SAXS, sf3636 may sample a range of relative domain orientations depending on data types and refinement tools. To further investigate sf3636 domain orientation and its intrinsic dynamic nature, Prestegard lab is developing a new protocol using RCSAs and RDCs from several mediums. Their refined models will be deposited later.	NMRPipe

NMR Ensemble Information
Conformer Selection Criteria	structures with the lowest energy
Conformers Calculated Total Number	100
Conformers Submitted Total Number	20
Representative Model	1 (lowest energy)

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	processing	NMRPipe	2.3	Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax
2	processing	MDDGUI	1.0	Gutmanas, Arrowsmith
3	data analysis	Sparky	3.95	Goddard
4	chemical shift assignment	FMCGUI	2.4	Lemak, Arrowsmith
5	structure solution	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
6	refinement	CNS		Brunger, Adams, Clore, Gros, Nilges and Read
7	nmr structure quality assessment	AutoStructure		Huang, Tejero, Powers and Montelione
8	nmr structure quality assessment	PSVS		Bhattacharya and Montelione
9	chemical shift assignment	ABACUS		Lemak, Arrowsmith

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.