2KKQ

Solution NMR Structure of the Ig-like C2-type 2 Domain of Human Myotilin. Northeast Structural Genomics Target HR3158.

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	3D CBCA(CO)NH	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
2	3D HNCA	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
3	3D HNCACB	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
4	3D HBHA(CO)NH	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
5	3D HCCH-COSY	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
6	3D CCH-TOCSY	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
7	3D 1H-15N NOESY	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
8	3D 1H-13C NOESY	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
9	3D HNCO	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
10	3D HN(CA)CO	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
11	2D 1H-15N HSQC	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
12	2D 1H-13C HSQC	0.87 mM [U-100% 13C; U-100% 15N] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
13	2D 1H-13C HSQC hires	0.68 mM [U-5% 13C] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
14	2D 1H-15N het_NOE	0.68 mM [U-5% 13C] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
15	1D 15N_T1 series	0.68 mM [U-5% 13C] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298
16	1D 15N_T2 series	0.68 mM [U-5% 13C] protein, 200 mM sodium chloride, 20 mM MES, 50 uM DSS, 10 mM DTT, 0.02 % sodium azide, 5 mM Calcium Chloride	90% H2O/10% D2O	0.2	6.5	ambient	298

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

NMR Refinement
Method	Details	Software
molecular dynamics	20 OF 100 STRUCTURES LOWEST TARGET FUNCTION SELECTED WITH CYANA-3.0. SELECTED MODELS ARE FURTHER REFINED USING CNS IN EXPLICIT WATER SHELL (NILGES PROTOCOL WITH PARAM19). STRUCTURE BASED ON 2412 NOE, 230 DIHE. MAX NOE VIOLATION: 0.23 A (1MODEL); MAX DIHE VIOLATION: 8.1 DEG. 3 TOTAL CLOSE CONTACTS PER 20 MODELS. STRUCTURE QUALITY FACTOR (PSVS 1.3): ORDERED RESIDUES RANGES: 11-112 FOR [S(PHI)+S(PSI)] > 1.8. SECONDARY STRUCTURE - BETA STRANDS: 25-28, 31-38, 46-51, 54-55, 63-67, 72-80, 86-94, 97-108 RMSD(ANG): BACKBONE 0.4, ALL HEAVY ATOMS 0.7. RAMA. DISTRIBUTION: 94.7/5.1/0.2/0.0. PROCHECK (PSI-PHI): -0.39/-1.22 (RAW/Z), PROCHECK (ALL): -0.23/-1.36 (RAW/Z), MOLPROBITY CLASH: 14.45/-1.01 (RAW/Z). RPF SCORES ALL ASSIGNED RESIDUES (FIT OF NOESY PEAKLISTS TO STRUCTURE): RECALL: 0.892, PRECISION: 0.934, F-MEASURE: 0.913, DP-SCORE: 0.76.	CYANA

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

Additional NMR Experimental Information
Details	MONOMER BY GEL FILTRATION CHROMATOGRAPHY/LIGHT SCATTERING AND BY NMR. T1/T2(CPMG). CONSISTENT WITH MOLECULAR WEIGHT OF MONOMERIC UNIT. STRUCTURE DETERMINED BY TRIPLE RESONANCE NMR SPECTROSCOPY. NOESY ASSIGNMENTS BY CYANA-3.0. ASSIGNMENT STATS (ALL RESIDUES INCLUDED): BACKBONE 90.16%, SIDECHAIN 76.91%, AROMATIC (SC) 100%, STEREOSPECIFIC VL METHYL ASSIGNMENT 100%, UNAMBIGUOUS SIDECHAIN NH2 100%.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	structure solution	CYANA	3.0	Guntert, Mumenthaler and Wuthrich
2	collection	TopSpin	2.1	Bruker Biospin
3	refinement	CNS	1.3	Brunger, Adams, Clore, Gros, Nilges and Read
4	data analysis	Sparky	2.113	Goddard
5	processing	NMRPipe		Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax
6	data analysis	PINE		Bahrami, Markley, Assadi, and Eghbalnia
7	structure visualization	MOLMOL		Koradi, Billeter and Wuthrich
8	structure validation	PSVS	1.3	Bhattacharya and Montelione
9	structure visualization	PyMOL		DeLano Scientific
10	pdb processing	PdbStat	5.1	Tejero, Montelione

RCSB PDB Core Operations are funded by the National Science Foundation (DBI-1832184), 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.