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BMRB: 26045

2ND2

Solution structure of the de novo mini protein HHH_06

Authors

Eletsky, A., Bahl, C.D., Gilmore, J.M., Buchko, G.W., Baker, D.

Assembly

HHH 06

Entity

1. HHH 06 (polymer, Thiol state: all disulfide bound), 44 monomers, 5119.811 Da Detail

APCEDLKERL KKLGMSEECR QRLEKMCKEG TSEDAERMAR NCES

Formula weight

5119.811 Da

Entity Connection

disulfide 2 Detail

ID	Type	Value order	Atom ID 1	Atom ID 2
1	disulfide	sing	1:CYS3:SG	1:CYS27:SG
2	disulfide	sing	1:CYS19:SG	1:CYS42:SG

Exptl. method

Refine. method

torsion angle dynamics, molecular dynamics

Data set

assigned_chemical_shifts, spectral_peak_list

Chem. Shift Complete

Sequence coverage: 100.0 %, Completeness: 93.1 %, Completeness (bb): 95.8 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	93.1 % (470 of 505)	97.1 % (266 of 274)	85.5 % (159 of 186)	100.0 % (45 of 45)
Backbone	95.8 % (251 of 262)	97.8 % (87 of 89)	93.1 % (121 of 130)	100.0 % (43 of 43)
Sidechain	89.1 % (254 of 285)	96.8 % (179 of 185)	74.5 % (73 of 98)	100.0 % (2 of 2)
Methyl	79.2 % (19 of 24)	100.0 % (12 of 12)	58.3 % (7 of 12)

1. HHH 06

APCEDLKERL KKLGMSEECR QRLEKMCKEG TSEDAERMAR NCES

Show sample condition

Sample

Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

Protein Blocks Logo

Calculated from 20 models in PDB: 2ND2, Strand ID: A Detail

Release date

2016-09-12

Citation

Accurate de novo design of hyperstable constrained peptides
Bhardwaj, G., Mulligan, V.K., Bahl, C.D., Gilmore, J.M., Harvey, P.J., Cheneval, O., Buchko, G.W., Pulavarti, S.V., Kaas, Q., Eletsky, A., Huang, P.S., Johnsen, W.A., Greisen, P.J., Rocklin, G.J., Song, Y., Linsky, T.W., Watkins, A., Rettie, S.A., Xu, X., Carter, L.P., Bonneau, R., Olson, J.M., Coutsias, E., Correnti, C.E., Szyperski, T., Craik, D.J., Baker, D.
Nature (2016), 538, 329-335, PubMed 27626386 , DOI 10.1038/nature19791 , Abstract

Related entities 1. HHH 06, : 4 entities Detail

Experiments performed 11 experiments Detail

1 2D 1H-15N HSQC

Instrument

Agilent DD2 - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

2 2D 1H-13C HSQC aliphatic

Instrument

Varian INOVA - 750 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

3 2D 1H-13C HSQC aromatic

Instrument

Varian INOVA - 750 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

4 2D 1H-1H DPFGSE TOCSY

Instrument

Varian INOVA - 750 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

5 2D 1H-1H DPFGSE NOESY

Instrument

Varian INOVA - 750 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

6 3D HNCO

Instrument

Varian INOVA - 750 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

7 3D HNCA

Instrument

Varian INOVA - 750 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

8 3D 1H-15N NOESY HSQC

Instrument

Varian INOVA - 750 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

9 3D 1H-15N TOCSY HSQC

Instrument

Varian INOVA - 750 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

10 3D HNHA

Instrument

Varian INOVA - 750 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 15N]		1 mM

11 3D HNHB

Instrument

Agilent DD2 - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100 % 15N] labeled HHH_06

#	Name	Isotope labeling	Type	Concentration
1	sodium phosphate	natural abundance		50 mM
2	DSS	natural abundance		4 uM
3	D2O	[U-99% 2H]		10 %
4	sodium azide	natural abundance		0.02 %
5	HHH_06	[U-100% 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_26045_2nd2.nef
Input source #2: Coordindates	2nd2.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:3:CYS:SG	A:27:CYS:SG	oxidized, CA 57.465, CB 39.971 ppm	oxidized, CA 59.277, CB 40.302 ppm	2.019
A:19:CYS:SG	A:42:CYS:SG	oxidized, CA 59.285, CB 40.375 ppm	oxidized, CA 57.045, CB 41.719 ppm	2.029

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----
APCEDLKERLKKLGMSEECRQRLEKMCKEGTSEDAERMARNCES
||||||||||||||||||||||||||||||||||||||||||||
APCEDLKERLKKLGMSEECRQRLEKMCKEGTSEDAERMARNCES

Chain assignments

Entity_assembly_ID (NMR)	Auth_asym_ID (model)	Length	Unmapped	Conflict	Sequence coverage (%)
A	A	44	0	0	100.0

Content subtype: combined_26045_2nd2.nef

#	Content subtype	Saveframe	Status	# of rows (sets)	Experiment type	Sequence coverage (%)
1	Covalent bonds	assembly	OK	2		No information
1	Assigned chemical shifts	assigned_chem_shift_list_1	Warning	477		100.0 (chain: A, length: 44)
1	Distance restraints	CNS/XPLOR_distance_constraints_2	OK	1125 (1)	noe	100.0 (chain: A, length: 44)
1	Dihedral angle restraints	CNS/XPLOR_dihedral_3	OK	54 (54)	.	75.0 (chain: A, length: 44)

Assigned chemical shifts

Saveframe: assigned_chem_shift_list_1
- Status: Warning
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 44, Sequence coverage: 100.0%
- Total number of assignments
  - ¹H chemical shifts: 273
  - ¹³C chemical shifts: 155
  - ¹⁵N chemical shifts: 49
- Completeness of assignments
  - Entity_assemble_ID: A
- Redox state of CYS
- 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: OK
- Experiment type: noe
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 44, Sequence coverage: 100.0%
- Total number of restraints: 1123
- Weight of restraints: 1.0 (1123)
- Potential type of restraints: square-well-parabolic (1123)
- Range of distance target values: 1.31, 2.89 (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: 44, Sequence coverage: 75.0%
- Total number of restraints: 54
- Total number of restraints per polymer type: 54
- Weight of restraints: 1.0 (54)
- Potential type of restraints: square-well-parabolic (54)
- Plot of Phi-Psi angle restraints
- Dihedral angle restraints per residue
  - Chain_ID: A

Keywords NMR, Solution structure, engineered protein