2KL7

Solution NMR Structure of the EGF-like 1 Domain of Human Fibulin-4. Northeast Structural Genomics Target HR6275

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	2D 1H-15N HSQC	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
2	2D 1H-13C HSQC	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
3	3D CBCA(CO)NH	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
4	3D HNCACB	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
5	3D HNCO	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
6	3D HBHA(CO)NH	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
7	3D HN(CA)CO	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
8	3D 1H-15N NOESY	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
9	3D 1H-13C NOESY	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
10	3D HCCH-TOCSY	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
11	3D HCCH-COSY	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
12	3D CCH-TOCSY	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
13	2D 1H-15N HMQC	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
14	2D 1H-15N het_NOE	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
15	1D 15N_T1 series	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
16	1D 15N_T2 series	0.6 mM [U-100% 13C; U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	6.5	ambient	298
17	2D 1H-15N HSQC	0.3 mM [U-100% 15N] protein, 150 mM sodium chloride, 20 mM MES, 50 uM DSS	90% H2O/10% D2O	0.15	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	NOESY ASSIGNMENTS BY CYANA-3.0. 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 776 NOE, 106 DIHE. MAX NOE VIOLATION: 0.23 A (1MODEL); MAX DIHE VIOLATION: 7.1 DEG. 1 TOTAL CLOSE CONTACTS PER 20 MODELS. STRUCTURE QUALITY FACTOR (PSVS 1.3): ORDERED RESIDUES RANGES: 11-34,53-66 (FINDCORE). SECONDARY STRUCTURE - BETA STRANDS: 17-21, 25-29, 61-63. RMSD(ANG): BACKBONE 1.0, ALL HEAVY ATOMS 1.3. RAMA. DISTRIBUTION: 94.1/5.9/0.0/0.0. PROCHECK (PSI-PHI): -0.38/-1.18 (RAW/Z), PROCHECK (ALL): -0.22/-1.30 (RAW/Z), MOLPROBITY CLASH: 16.14/-1.24 (RAW/Z). RPF SCORES ALL ASSIGNED RESIDUES (FIT OF NOESY PEAKLISTS TO STRUCTURE): RECALL: 0.880, PRECISION: 0.916, F-MEASURE: 0.898, DP-SCORE: 0.651.	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) T1=664.9 MS T2=128.48 MS TAUC=4.8 NS. CONSISTENT WITH MOLECULAR WEIGHT OF MONOMERIC UNIT. STRUCTURE DETERMINED BY TRIPLE RESONANCE NMR SPECTROSCOPY. ASSIGNMENT STATS (ALL RESIDUES INCLUDED): BACKBONE 91.89%, SIDECHAIN 83.74%, AROMATIC (SC) 100%. UNAMBIGUOUS SIDECHAIN NH2 100%.

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

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.