Experiment: 2M4E

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	3D HNCO	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
2	3D CBCA(CO)NH	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
3	3D HBHA(CO)NH	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
4	3D HNCA	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
5	3D HCCH-TOCSY	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
6	3D CCH-TOCSY	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
7	3D 1H-15N NOESY	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
8	3D 1H-13C NOESY	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
9	3D 1H-13C NOESY aromatic	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
10	2D 1H-13C HSQC	0.2 mM [U-7% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
11	3D HNCACB	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
12	3D HNCB	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
13	3D C(CO)NH	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
14	3D H(CCO)NH	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
15	3D HCCH-COSY	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298
16	3D TOCSY-HSQC	0.5 mM [U-100% 13C; U-100% 15N] VV2_0175, 10 mM [U-100% 2H] TRIS, 300 mM sodium chloride, 10 mM [U-100% 2H] DTT, 0.01 % NaN3, 10 mM benzamidine, 1 % inhibitor cocktail	90% H2O/10% D2O	300	7.0	ambient	298

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	AVANCE	800
2	Bruker	AVANCE	600
3	Bruker	AVANCE	500

NMR Refinement
Method	Details	Software
simulated annealing	The heterogeneity of viv0002 expressed in Escherichia coli BL21 (DE3) was observed. Three extra peaks in 15N HSQC and more than 25 extra peaks in 13C HSQC were identified. These peaks are grouped into three fragments, one assigned as GlcNAc, the other two fragments could not be accurately assigned given the limited information we have, but they are likely to belong to glycans. These fragments display intra NOEs only and remain solvent exposed with strong and narrow line widths. NMR relaxation data showed that these unknown modifications are highly mobile and no interaction with viv0002. Heterologously expressed viv0002 was confirmed by LC-MS. A mass shift of 178 Da of 15N-labeled viv0002 was detected whereas a shift of 72 Da for 13C/15N-labeled sample. However, neither of results matches any three unknown fragments seen in the NMR spectrum with MW 202 Da, 155 Da and 204 Da, respectively. The nature and functional consequence of the unknown fragments/modifications to viv0002 are not understood at this stage. The structure deposited provides the basis of the future investigation.	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	2.1	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	FAWN		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.