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BMRB: 30314

5WBT

Solution Structure and Dynamics of an Ultra-Stable Single-Chain Insulin Analog STUDIES OF AN ENGINEERED MONOMER AND IMPLICATIONS FOR RECEPTOR BINDING

Authors

Glidden, M.D., Yang, Y., Wickramasinghe, N.P., Weiss, M.A.

Assembly

Insulin

Entity

1. Insulin (polymer), 57 monomers, 6509.257 Da Detail

FVNQHLCGSH LVEALYLVCG ERGFFYTDPT EEGPRRGIVE QCCHSICSLE QLENYCN

Formula weight

6509.257 Da

Source organism

Exptl. method

Refine. method

simulated annealing

Data set

assigned_chemical_shifts

Chem. Shift Complete

Sequence coverage: 98.2 %, Completeness: 76.9 %, Completeness (bb): 83.4 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	76.9 % (500 of 650)	73.2 % (246 of 336)	81.4 % (206 of 253)	78.7 % (48 of 61)
Backbone	83.4 % (282 of 338)	87.2 % (102 of 117)	83.1 % (138 of 166)	76.4 % (42 of 55)
Sidechain	72.5 % (264 of 364)	65.8 % (144 of 219)	82.0 % (114 of 139)	100.0 % (6 of 6)
Aromatic	25.8 % (17 of 66)	9.1 % (3 of 33)	42.4 % (14 of 33)
Methyl	100.0 % (54 of 54)	100.0 % (27 of 27)	100.0 % (27 of 27)

1. Insulin

FVNQHLCGSH LVEALYLVCG ERGFFYTDPT EEGPRRGIVE QCCHSICSLE QLENYCN

Show sample condition

Sample

Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

LACS Plot; CA

Referencing offset: -0.11 ppm, Outliers: 4 Detail

LACS Plot; CB

Referencing offset: -0.11 ppm, Outliers: 4 Detail

LACS Plot; HA

Referencing offset: -0.02 ppm, Outliers: 1 Detail

LACS Plot; CO

Referencing offset: 0.17 ppm, Outliers: 2 Detail

Protein Blocks Logo

Calculated from 19 models in PDB: 5WBT, Strand ID: A Detail

Release date

2017-07-20

Citation

Solution structure of an ultra-stable single-chain insulin analog connects protein dynamics to a novel mechanism of receptor binding
Glidden, M.D., Yang, Y., Smith, N.A., Phillips, N.B., Carr, K., Wickramasinghe, N.P., Ismail-Beigi, F., Lawrence, M.C., Smith, B.J., Weiss, M.A.
J. Biol. Chem. (2017), 293, 69-88, PubMed 29114034 , DOI 10.1074/jbc.M117.808667 , Abstract

Related entities 1. Insulin, : 1 : 1 : 3 : 156 entities Detail

Experiments performed 13 experiments Detail

1 4D Time-shared NOESY

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

2 3D HNCA

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

3 3D CBCA(CO)NH

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

4 3D HNCO

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

5 3D HNCACB

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

6 3D HN(CO)CA

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

7 3D 1H-15N TOCSY

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

8 3D H(CCO)NH

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

9 3D HCC-TOCSY

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

10 2D 1H-13C HSQC aromatic

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

11 2D 1H-13C HSQC

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

12 2D 1H-15N HSQC

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

13 3D (H)CC(CO)NH

Instrument

Bruker Avance - 700 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 7.4, Details 1 mM [U-13C; U-15N] Single chain insulin SCI-b, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	Single chain insulin SCI-b	[U-13C; U-15N]		1 mM

NMR combined restraints 4 contents Detail

Properties

Input source #1: NMR data (NEF) - Assigned chemical shifts, Distance restraints, Dihedral angle restraints	combined_30314_5wbt.nef
Input source #2: Coordindates	5wbt.cif
Diamagnetism of the molecular assembly	True (excluding Oxygen atoms)
Whether the assembly has a disulfide bond	True (see coordinates for details)
Whether the assembly has a other bond	None
Whether the assembly contains a cyclic polymer	None
Overall data processing status	Warning

Disulfide bonds

Ptnr_site_1	Ptnr_site_2	Redox_state_prediction_1	Redox_state_prediction_2	Distance (Å)
A:7:CYS:SG	A:43:CYS:SG	unknown	unknown	2.022
A:19:CYS:SG	A:56:CYS:SG	unknown	unknown	2.024
A:42:CYS:SG	A:47:CYS:SG	unknown	unknown	2.025

Other bonds (neither disulfide, covalent nor hydrogen bonds, e.g. Zinc–sulphur bond)

None

Non-standard residues

None

Sequence alignments

Entity_assembly_ID (NMR): A vs Auth_asym_ID (model): A

--------10--------20--------30--------40--------50-------
FVNQHLCGSHLVEALYLVCGERGFFYTDPTEEGPRRGIVEQCCHSICSLEQLENYCN
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||
FVNQHLCGSHLVEALYLVCGERGFFYTDPTEEGPRRGIVEQCCHSICSLEQLENYCN

Chain assignments

Entity_assembly_ID (NMR)	Auth_asym_ID (model)	Length	Unmapped	Conflict	Sequence coverage (%)
A	A	57	0	0	100.0

Content subtype: combined_30314_5wbt.nef

#	Content subtype	Saveframe	Status	# of rows (sets)	Experiment type	Sequence coverage (%)
1	Covalent bonds	assembly	OK	3		No information
1	Assigned chemical shifts	assigned_chemical_shifts_1	Warning	498		98.2 (chain: A, length: 57)
1	Distance restraints	CNS/XPLOR_distance_constraints_2	Warning	1709 (1)	noe	96.5 (chain: A, length: 57)
1	Dihedral angle restraints	CNS/XPLOR_dihedral_3	OK	76 (76)	.	82.5 (chain: A, length: 57)

Assigned chemical shifts

Saveframe: assigned_chemical_shifts_1
- Status: Warning
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 57, Sequence coverage: 98.2%
- Total number of assignments
  - ¹H chemical shifts: 245
  - ¹³C chemical shifts: 205
  - ¹⁵N chemical shifts: 48
- Completeness of assignments
  - Entity_assemble_ID: A
- Peptide bond of PRO
- Rotameric state of ILE , LEU, and VAL
- Histogram of normalized assigned chemical shifts
- Random coil index and derived parameters (S² and NMR RMSD)
  - Chain_ID: A

Covalent bonds

Saveframe: assembly
- Status: OK

Distance restraints

Saveframe: CNS/XPLOR_distance_constraints_2
- Status: Warning
- Experiment type: noe
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 57, Sequence coverage: 96.5%
- Total number of restraints: 1692
- Weight of restraints: 1.0 (1692)
- Potential type of restraints: square-well-parabolic (1692)
- Range of distance target values: 2.45, 3.9 (Å)
- Histogram of distance target values
- Histogram of discrepancy in distance target values
- Distance restraints per residue
  - Chain_ID: A
- Distance restraints on contact map

Dihedral angle restraints

Saveframe: CNS/XPLOR_dihedral_3
- Status: OK
- Experiment type: .
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 57, Sequence coverage: 82.5%
- Total number of restraints: 76
- Total number of restraints per polymer type: 76
- Weight of restraints: 1.0 (76)
- Potential type of restraints: square-well-parabolic (76)
- Plot of Phi-Psi angle restraints
- Dihedral angle restraints per residue
  - Chain_ID: A

Keywords HORMONE, heat-stable