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BMRB: 26046

2ND3

Solution structure of the de novo mini protein EEH_04

Authors

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

Assembly

EEH 04

Entity

1. EEH 04 (polymer, Thiol state: all disulfide bound), 38 monomers, 4655.142 Da Detail

QCYTFRSECT NKEFTVCRPN PEEVEKEARR TKEEECRK

Formula weight

4655.142 Da

Entity Connection

disulfide 2 Detail

ID	Type	Value order	Atom ID 1	Atom ID 2
1	disulfide	sing	1:CYS2:SG	1:CYS17:SG
2	disulfide	sing	1:CYS9:SG	1:CYS36:SG

Exptl. method

Refine. method

distance geometry, molecular dynamics

Data set

assigned_chemical_shifts, spectral_peak_list

Chem. Shift Complete

Sequence coverage: 100.0 %, Completeness: 82.6 %, Completeness (bb): 86.6 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	82.6 % (379 of 459)	92.7 % (230 of 248)	64.5 % (111 of 172)	97.4 % (38 of 39)
Backbone	86.6 % (194 of 224)	98.6 % (73 of 74)	75.4 % (86 of 114)	97.2 % (35 of 36)
Sidechain	72.9 % (199 of 273)	90.2 % (157 of 174)	40.6 % (39 of 96)	100.0 % (3 of 3)
Aromatic	100.0 % (28 of 28)	100.0 % (14 of 14)	100.0 % (14 of 14)
Methyl	100.0 % (18 of 18)	100.0 % (9 of 9)	100.0 % (9 of 9)

1. entity

QCYTFRSECT NKEFTVCRPN PEEVEKEARR TKEEECRK

Show sample condition

Sample

Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

Protein Blocks Logo

Calculated from 20 models in PDB: 2ND3, 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. EEH 04, : 1 entities Detail

Experiments performed 13 experiments Detail

1 2D 1H-15N HSQC

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

2 2D 1H-13C HSQC aliphatic

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

3 2D 1H-13C HSQC aromatic

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

4 2D 1H-13C HSQC aliphatic ct

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,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

5 2D 1H-13C HSQC aliphatic ct

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,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

6 2D 1H-1H DPFGSE TOCSY

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

7 2D 1H-1H DPFGSE NOESY

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

8 3D HNHA

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

9 3D HNCO

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

10 3D HNCA

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

11 3D HNHB

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

12 3D 1H-15N NOESY HSQC

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

13 3D 1H-15N TOCSY HSQC

Instrument

Varian INOVA - 750 MHz Equuipped with cryogenic probe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0, Details [U-100% 15N,U-10% 13C] labeled EEH_04

#	Name	Isotope labeling	Type	Concentration
1	EEH_04	[U-10% 13C; U-100% 15N]		1 mM
2	sodium phosphate	natural abundance		50 mM
3	DSS	natural abundance		4 uM
4	D2O	[U-99% 2H]		10 %
5	sodium azide	natural abundance		0.02 %

NMR combined restraints 4 contents Detail

Properties

Input source #1: NMR data (NEF) - Assigned chemical shifts, Distance restraints, Dihedral angle restraints	combined_26046_2nd3.nef
Input source #2: Coordindates	2nd3.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:2:CYS:SG	A:17:CYS:SG	oxidized, CA 55.702, CB 41.876 ppm	oxidized, CA 54.966, CB 38.222 ppm	2.031
A:9:CYS:SG	A:36:CYS:SG	unknown, CA 58.379 ppm	unknown, CA 55.849 ppm	2.034

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--------
QCYTFRSECTNKEFTVCRPNPEEVEKEARRTKEEECRK
||||||||||||||||||||||||||||||||||||||
QCYTFRSECTNKEFTVCRPNPEEVEKEARRTKEEECRK

Chain assignments

Entity_assembly_ID (NMR)	Auth_asym_ID (model)	Length	Unmapped	Conflict	Sequence coverage (%)
A	A	38	0	0	100.0

Content subtype: combined_26046_2nd3.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	381		100.0 (chain: A, length: 38)
1	Distance restraints	CNS/XPLOR_distance_constraints_3	OK	833 (1)	noe	100.0 (chain: A, length: 38)
1	Dihedral angle restraints	CNS/XPLOR_dihedral_2	OK	44 (44)	.	73.7 (chain: A, length: 38)

Assigned chemical shifts

Saveframe: assigned_chem_shift_list_1
- Status: Warning
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 38, Sequence coverage: 100.0%
- Total number of assignments
  - ¹H chemical shifts: 229
  - ¹³C chemical shifts: 109
  - ¹⁵N chemical shifts: 43
- Completeness of assignments
  - Entity_assemble_ID: A
- Redox state of CYS
- 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_3
- Status: OK
- Experiment type: noe
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 38, Sequence coverage: 100.0%
- Total number of restraints: 830
- Weight of restraints: 1.0 (830)
- Potential type of restraints: square-well-parabolic (830)
- Range of distance target values: 1.1, 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_2
- Status: OK
- Experiment type: .
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 38, Sequence coverage: 73.7%
- Total number of restraints: 44
- Total number of restraints per polymer type: 44
- Weight of restraints: 1.0 (44)
- Potential type of restraints: square-well-parabolic (44)
- Plot of Phi-Psi angle restraints
- Dihedral angle restraints per residue
  - Chain_ID: A

Keywords NMR, Solution structure, engineered protein