Mass: 16286.207 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: Gene amplified from purified eosinophil RNA by RT-PCR. Source: (gene. exp.) Homo sapiens (human) / Cell: human blood eosinophils / Gene: MYDGF, C19orf10 / Plasmid: pET-ELMER Details (production host): pET28c(+) derivative (kanamycin resistance). Expresses proteins with N-terminal polyhistidine-tag followed by a thrombin cleavage site. Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2(DE3) / Variant (production host): MilliporeSigma 71400 / References: UniProt: Q969H8
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Experimental details
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Experiment
Experiment
Method: SOLUTION NMR
NMR experiment
Conditions-ID
Experiment-ID
Solution-ID
Sample state
Spectrometer-ID
Type
1
1
1
isotropic
1
3D 1H,15N-HSQCNOESY
1
2
1
isotropic
1
3D 1H,13C-HSQCNOESY aliphatic
1
3
1
isotropic
1
3D 1H,13C-HSQCNOESY aromatic
1
4
1
isotropic
2
2D 1H-15N HSQC
1
5
1
isotropic
2
2D 1H-13C HSQC aliphatic
1
6
1
isotropic
1
2D 1H-13C HSQC aromatic
1
7
1
isotropic
2
3DCBCA(CO)NH
1
8
1
isotropic
2
3D HN(CA)CB
1
9
1
isotropic
2
3D HNCO
1
10
1
isotropic
2
3DC(CO)NH
1
11
1
isotropic
2
3DHBHA(CO)NH
1
12
1
isotropic
2
3D (H)CCH-TOCSY aliphatic
1
13
1
isotropic
2
3DH(CCO)NH
1
14
1
isotropic
1
2D (HB)CB(CGCD)HDaromatic
1
15
1
isotropic
1
2D (HB)CB(CGCDCE)HDHEaromatic
1
16
1
isotropic
4
3D (H)CCH-TOCSY aromatic
1
20
1
isotropic
3
3D 1H,15N-HSQCNOESY 1H,13C-HSQC
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Sample preparation
Details
Type: solution Contents: 8.25 mg/mL U-15N; U-13C; NA-H MYDGF, 9 mM NA sodium phosphate, 135 mM NA sodium chloride, 15 uM NA DSS, 0.02 % w/v NA sodium azide, 90% H2O/10% D2O Details: DSS was used as an internal NMR standard and sodium azide was added to prevent contamination growth. Label: 15N_13C_MYDGF / Solvent system: 90% H2O/10% D2O
Sample
Conc. (mg/ml)
Component
Isotopic labeling
Solution-ID
8.25mg/mL
MYDGF
U-15N; U-13C; NA-H
1
9mM
sodiumphosphate
NA
1
135mM
sodiumchloride
NA
1
15uM
DSS
NA
1
0.02 % w/v
sodiumazide
NA
1
Sample conditions
Ionic strength: 135 (NaCl) mM / Label: Condition_1 / pH: 6 / Pressure: 1 atm / Temperature: 298 K
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NMR measurement
NMR spectrometer
Type
Manufacturer
Model
Field strength (MHz)
Spectrometer-ID
Varian VNS
Varian
VNS
800
1
Bruker AVANCE III
Bruker
AVANCEIII
600
2
Bruker AVANCE III
Bruker
AVANCEIII
900
3
Varian VNS
Varian
VNS
600
4
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Processing
NMR software
Name
Version
Developer
Classification
VNMR
4.2
Varian
collection
TopSpin
3.5
BrukerBiospin
collection
NMRPipe
Delaglio, Grzesiek, Vuister, Zhu, PfeiferandBax
processing
NMRFAM-SPARKY
W. Lee, M. TonelliandJ.L. Markley
chemicalshiftassignment
I-PINE
W. Lee, A. Bahrami, H. Dashti, H.R. Eghbalnia, M. Tonelli, W.M. Westler, J.L. Markley
chemicalshiftassignment
NMRFAM-SPARKY
W. Lee, M. TonelliandJ.L. Markley
peakpicking
PONDEROSA-C/S
W. Lee, J.L. Stark, J.L. Markley
structurecalculation
TALOS-N
ShenandBax
structurecalculation
X-PLOR NIH
Schwieters, Kuszewski, TjandraandClore
structurecalculation
PyMOL
Schroedinger, Inc.
refinement
PONDEROSA-C/S
W. Lee, J.L. Stark, J.L. Markley
refinement
X-PLOR NIH
Schwieters, Kuszewski, TjandraandClore
refinement
Refinement
Method
Software ordinal
Details
molecular dynamics
11
explicitwaterrefinement
molecular dynamics
12
explicitwaterrefinement
NMR representative
Selection criteria: lowest energy
NMR ensemble
Conformer selection criteria: all calculated structures submitted Conformers calculated total number: 100 / Conformers submitted total number: 20
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