1HYK

AGOUTI-RELATED PROTEIN (87-132) (AC-AGRP(87-132))


SOLUTION NMR
NMR Experiment
ExperimentTypeSample ContentsSolventIonic StrengthpHPressureTemperature (K)Spectrometer
12D-NOESY1.9 MM Ac-AGRP(87-132) (same as 1.9 MM MARP of 1qu8)50 MM PHOSPHATE BUFFER PH 5.0 (pH is changed from pH=4.2 in 1qu8)50mM PHOSPHATE5.001 atm288.00
2DQF-COSY1.9 MM Ac-AGRP(87-132) (same as 1.9 MM MARP of 1qu8)50 MM PHOSPHATE BUFFER PH 5.0 (pH is changed from pH=4.2 in 1qu8)50mM PHOSPHATE5.001 atm288.00
3E-COSY1.9 MM Ac-AGRP(87-132) (same as 1.9 MM MARP of 1qu8)50 MM PHOSPHATE BUFFER PH 5.0 (pH is changed from pH=4.2 in 1qu8)50mM PHOSPHATE5.001 atm288.00
NMR Spectrometer Information
SpectrometerManufacturerModelField Strength
1VarianUNITYPLUS800
NMR Refinement
MethodDetailsSoftware
torsion angle dynamicsThe preliminary structure calculation and restraint quality assessment was performed in DYANA v 1.5, so that out of 200 structures the 20 with the lowest target functions would have target function vaules of < 3.0 square angstroms. The lowest target function structure of this family was then submitted as a pre-folded structure in CNS v. 1.0, and 40 structures calculated under cool (< 288K) simulated annealing and conjugate gradient energy minimization. These calculations were carried out in the presence of experimnetal NOE and J-coupling restraints as well as assigned hydrogen bonds consistent with HX protection. DYANA 1.5: 799 unique NOE's from 800 MHz data, 444 resulting distance restraints after pseudoatom calculation/replacement and removal of NOE's between atoms separated by only two or three bonds. 35 alpha-H to amide-H 3-bond J-coupling constants and 44 alpha-H to beta-H 3-bond J-coupling constants from ECOSY data were also included. Disulfides and seven hydrogen bonds as determinied from surrounding NOE's and HX data were included as pseudo-NOE restraints. CNS 1.0, patch level 0: 504 distance restraints from 800 MHz NOE data, corresponding to the same upper limit restraints as used in the final round of DYANA 1.5 calulations (conversion using AQUA 2.0 in PROCHECK_NMR, and modified lower limit of 1.6 angstroms was imposed on all NOE restraints while keeping upper bounds). Disulfide map was incorporated into the covalent structure of the molecule and thereby strictly enforced in this step. The seven hydrogen bonds were included in a separate restraint file. 34 alpha-H to amide-H 3-bond J-coupling constants (not including CYS-87 amide mesurement) were also included. 40 structures were so calculated and all are deposited here.CNS
NMR Ensemble Information
Conformer Selection Criteriasee refinement method and details
Conformers Calculated Total Number40
Conformers Submitted Total Number40
Representative Model10 (lowest energy)
Additional NMR Experimental Information
DetailsSTANDARD 2D HOMONUCLEAR TECHNIQUES WERE USED TO ASSIGN SPECTRA ACQUIRED OVER THE TEMPERATURE RANGE 288- 303 K. A SUBSET OF UNAMBIGUOUSLY ASSIGNED NOE PEAKS TAKEN FROM DATA ACQUIRED AT 288 K WITH AN 150 MS MIXING TIME WERE USED IN STRUCTURE CALCULATIONS, AS WERE 34 PHI BACKBONE ANGLE RESTRAINTS DERIVED FROM FITTING OF DQF- COSY CROSSPEAKS. BOTH CARRIER PRESATURATION AND Z- GRADIENT WET TECHNIQUES WERE USED FOR SOLVENT SUPPRESSION. New experiments from 1qu8 are E-COSY at 500 MHz and 150 msec 2D-NOESY at 800 MHz, 288K.
Computation: NMR Software
#ClassificationVersionSoftware NameAuthor
1refinementCNS1.0A.T.BRUNGER, P.D.ADAMS, G.M.CLORE, W.L.DELANO, P.GROS, R.W.GROSSE-KUNSTLEVE, J.-S.JIANG, J.KUSZEWSKI, M.NILGES, N.S.PANNU, R.J.READ, L.M.RICE, T.SIMONSON, G.L.WARREN
2collectionVNMR5.2F. Vosman, D. Iverson, S. Patt, S. Chetham, R. Lasater, P. Hornung, G. Brissey, E. Williams, B. John, C. H. Yoder, B. L. Buckwalter
3processingMNMR940501M. Kjear, K.V. Andersoen, C. Rischel
4data analysisXEASY1.2T. Xia, C. Bartels
5structure solutionDYANA1.5P. Guntert, C. Mumenthaler, T. Herrmann
6processingPROCHECK3.4.4Laskowski R. A., MacArthur M. W., Moss D. S., Thornton J. M., Rullmann J. A. C.