2KJ1

cytoplasmic domain structure of BM2 proton channel from influenza B virus


SOLUTION NMR
NMR Experiment
ExperimentTypeSample ContentsSolventIonic StrengthpHPressureTemperature (K)Spectrometer
12D 1H-15N HSQC0.8 mM [U-100% 13C; U-100% 15N] cytoplasmic domain of BM2, 150 mM [U-100% 2H] LMPG, 25 mM sodium chloride, 25 mM TRIS93% H2O/7% D2O256.8ambient305
23D 1H-15N NOESY0.8 mM [U-100% 15N] cytoplasmic domain of BM2, 150 mM LMPG, 25 mM sodium chloride, 25 mM TRIS93% H2O/7% D2O256.8ambient305
33D 1H-13C NOESY0.8 mM [U-100% 15N] cytoplasmic domain of BM2, 150 mM LMPG, 25 mM sodium chloride, 25 mM TRIS93% H2O/7% D2O256.8ambient305
43D HNCA0.8 mM [U-100% 13C; U-100% 15N; U-80% 2H] cytoplasmic domain of BM2, 150 mM LMPG, 25 mM sodium chloride, 25 mM TRIS93% H2O/7% D2O256.8ambient305
53D HNCACB0.8 mM [U-100% 13C; U-100% 15N; U-80% 2H] cytoplasmic domain of BM2, 150 mM LMPG, 25 mM sodium chloride, 25 mM TRIS93% H2O/7% D2O256.8ambient305
63D HNCO0.8 mM [U-100% 13C; U-100% 15N; U-80% 2H] cytoplasmic domain of BM2, 150 mM LMPG, 25 mM sodium chloride, 25 mM TRIS93% H2O/7% D2O256.8ambient305
72D 1H-13C HSQC0.8 mM [U-100% 13C; U-100% 15N; U-80% 2H] cytoplasmic domain of BM2, 150 mM LMPG, 25 mM sodium chloride, 25 mM TRIS93% H2O/7% D2O256.8ambient305
8J(NCgamma-aliphatic)0.8 mM [U-100% 15N] cytoplasmic domain of BM2, 150 mM LMPG, 25 mM sodium chloride, 25 mM TRIS93% H2O/7% D2O256.8ambient305
9J-scaled TROSY-HNCO0.5 mM [U-10% 13C] cytoplasmic domain of BM2, 150 mM potassium form of LMPG, 25 mM potassium chloride, 25 mM TRIS, 2.9 % dGpdG93% H2O/7% D2O256.8ambient305
103D (1H-13C-HMQC)-NOESY-(1H-15N-TROSY)0.8 mM [ILV_methyl_13C; U-100% 15N; U-80% 2H] cytoplasmic domain of BM2, 150 mM [U-100% 2H] LMPG, 25 mM sodium chloride, 25 mM TRIS93% H2O/7% D2O256.8ambient305
113D (1H-13C-HMQC)-NOESY-(1H-13C-HSQC)0.8 mM [ILV_methyl_13C; U-100% 15N; U-80% 2H] cytoplasmic domain of BM2, 150 mM [U-100% 2H] LMPG, 25 mM sodium chloride, 25 mM TRIS93% H2O/7% D2O256.8ambient305
NMR Spectrometer Information
SpectrometerManufacturerModelField Strength
1BrukerAVANCE600
2BrukerAVANCE750
3BrukerAVANCE900
NMR Refinement
MethodDetailsSoftware
simulated annealingStructures were calculated using the program XPLOR-NIH (Schwieters et al., 2002). The secondary structure of the monomer was first calculated from random coil using intra-subunit NOEs, backbone dihedral restraints derived from chemical shifts (TALOS) (Cornilescu et al., 1999), and side chain χ1 restraints. A total of 20 monomer structures were calculated using a standard high-temperature simulated annealing (SA) protocol in which the bath temperature was cooled from 1000 to 200 K. To obtain an initial set of tetramer structures, four copies of the lowest-energy monomer structure calculated above were used. The same SA run was performed in the presence of inter-subunit NOEs and all other intra-subunit restraints. For each experimental inter-subunit NOE between two adjacent subunits, four identical distance restraints were assigned respectively to all pairs of neighboring subunits to satisfy the condition of C4 rotational symmetry. During the annealing run, the bath was cooled from 1000 to 200 K with a temperature step of 20 K. A total of 100 tetramer structures were calculated. For BM2(1-33), 15 low energy structures were selected as the structural ensemble.X-PLOR NIH
NMR Ensemble Information
Conformer Selection Criteriastructures with the lowest energy
Conformers Calculated Total Number100
Conformers Submitted Total Number15
Representative Model1 (lowest energy)
Computation: NMR Software
#ClassificationVersionSoftware NameAuthor
1refinementX-PLOR NIHSchwieters, C.D. et al.
2processingNMRPipeDelaglio, F. et al.
3data analysisNMRDrawDelaglio, F. et al.
4collectionXwinNMRBruker Biospin
5chemical shift calculationTALOSCornilescu, G. et al.
6structure solutionProcheckNMRLaskowski, R. et al.
7chemical shift assignmentCARAKeller, R. et al.