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BMRB: 30164

5T4R

NMR solution structure of the Nav1.7 selective spider venom-derived peptide Pn3a

Authors

Rosengren, K.J., Armstrong, D.A., Vetter, I.

Assembly

Mu-theraphotoxin-Pn3a

Entity

1. Mu-theraphotoxin-Pn3a (polymer), 35 monomers, 4277.923 Da Detail

DCRYMFGDCE KDEDCCKHLG CKRKMKYCAW DFTFT

Formula weight

4277.923 Da

Source organism

Exptl. method

Refine. method

simulated annealing

Data set

assigned_chemical_shifts

Chem. Shift Complete

Sequence coverage: 100.0 %, Completeness: 73.8 %, Completeness (bb): 82.4 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	73.8 % (310 of 420)	87.8 % (194 of 221)	50.9 % (83 of 163)	91.7 % (33 of 36)
Backbone	82.4 % (173 of 210)	98.6 % (71 of 72)	67.0 % (69 of 103)	94.3 % (33 of 35)
Sidechain	69.5 % (169 of 243)	82.6 % (123 of 149)	49.5 % (46 of 93)	0.0 % (0 of 1)
Aromatic	37.1 % (23 of 62)	74.2 % (23 of 31)	0.0 % (0 of 30)	0.0 % (0 of 1)
Methyl	60.0 % (6 of 10)	100.0 % (5 of 5)	20.0 % (1 of 5)

1. entity 1

DCRYMFGDCE KDEDCCKHLG CKRKMKYCAW DFTFT

Show sample condition

Sample #1

Solvent system 90% H2O/10% D2O, Pressure 1 null, Temperature 298 K, pH 4.0, Details 2 mg/mL mu-theraphotoxin-Pn3a, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	mu-theraphotoxin-Pn3a	natural abundance		2 (±0.2) mg/mL

Sample #2

Solvent system 100% D2O, Pressure 1 null, Temperature 298 K, pH 4.0, Details 2 mg/mL mu-theraphotoxin-Pn3a, 100% D2O

#	Name	Isotope labeling	Type	Concentration
2	mu-theraphotoxin-Pn3a	natural abundance		2 (±0.2) mg/mL

LACS Plot; CA

Referencing offset: -0.03 ppm, Outliers: 1 Detail

LACS Plot; CB

Referencing offset: -0.03 ppm, Outliers: 1 Detail

LACS Plot; HA

Referencing offset: 0.07 ppm, Outliers: 1 Detail

Protein Blocks Logo

Calculated from 20 models in PDB: 5T4R, Strand ID: A Detail

Release date

2016-11-10

Citation

Recombinant production, bioconjugation and membrane binding studies ofPn3a, a selective Na_V1.7 inhibitor
Sharma, G., Deuis, J.R., Jia, X., Mueller, A., Vetter, I., Mobli, M.
Biochem. Pharmacol. (2020), 181, 114148-114148, PubMed 32663452 , DOI 10.1016/j.bcp.2020.114148 , Abstract

Related entities 1. Mu-theraphotoxin-Pn3a, : 1 : 117 entities Detail

Experiments performed 7 experiments Detail

1 2D 1H-1H NOESY

Instrument

Bruker Avance - 600 MHz equipped with cryoprobe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 null, Temperature 298 K, pH 4.0, Details 2 mg/mL mu-theraphotoxin-Pn3a, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	mu-theraphotoxin-Pn3a	natural abundance		2 (±0.2) mg/mL

2 2D 1H-1H TOCSY

Instrument

Bruker Avance - 600 MHz equipped with cryoprobe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 null, Temperature 298 K, pH 4.0, Details 2 mg/mL mu-theraphotoxin-Pn3a, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	mu-theraphotoxin-Pn3a	natural abundance		2 (±0.2) mg/mL

3 2D 1H-15N HSQC

Instrument

Bruker Avance - 600 MHz equipped with cryoprobe

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 null, Temperature 298 K, pH 4.0, Details 2 mg/mL mu-theraphotoxin-Pn3a, 90% H2O/10% D2O

#	Name	Isotope labeling	Type	Concentration
1	mu-theraphotoxin-Pn3a	natural abundance		2 (±0.2) mg/mL

4 2D 1H-1H NOESY

Instrument

Bruker Avance - 600 MHz equipped with cryoprobe

Sample

State isotropic, Solvent system 100% D2O, Pressure 1 null, Temperature 298 K, pH 4.0, Details 2 mg/mL mu-theraphotoxin-Pn3a, 100% D2O

#	Name	Isotope labeling	Type	Concentration
2	mu-theraphotoxin-Pn3a	natural abundance		2 (±0.2) mg/mL

5 2D 1H-1H TOCSY

Instrument

Bruker Avance - 600 MHz equipped with cryoprobe

Sample

State isotropic, Solvent system 100% D2O, Pressure 1 null, Temperature 298 K, pH 4.0, Details 2 mg/mL mu-theraphotoxin-Pn3a, 100% D2O

#	Name	Isotope labeling	Type	Concentration
2	mu-theraphotoxin-Pn3a	natural abundance		2 (±0.2) mg/mL

6 2D 1H-13C HSQC

Instrument

Bruker Avance - 600 MHz equipped with cryoprobe

Sample

State isotropic, Solvent system 100% D2O, Pressure 1 null, Temperature 298 K, pH 4.0, Details 2 mg/mL mu-theraphotoxin-Pn3a, 100% D2O

#	Name	Isotope labeling	Type	Concentration
2	mu-theraphotoxin-Pn3a	natural abundance		2 (±0.2) mg/mL

7 2D 1H-1H ECOSY

Instrument

Bruker Avance - 600 MHz equipped with cryoprobe

Sample

State isotropic, Solvent system 100% D2O, Pressure 1 null, Temperature 298 K, pH 4.0, Details 2 mg/mL mu-theraphotoxin-Pn3a, 100% D2O

#	Name	Isotope labeling	Type	Concentration
2	mu-theraphotoxin-Pn3a	natural abundance		2 (±0.2) mg/mL

NMR combined restraints 5 contents Detail

Properties

Input source #1: NMR data (NEF) - Assigned chemical shifts, Distance restraints, Dihedral angle restraints	combined_30164_5t4r.nef
Input source #2: Coordindates	5t4r.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:16:CYS:SG	oxidized, CA 54.571, CB 41.488 ppm	oxidized, CA 53.223, CB 38.962 ppm	2.034
A:9:CYS:SG	A:21:CYS:SG	oxidized, CA 54.109, CB 46.852 ppm	oxidized, CA 54.69, CB 39.334 ppm	2.029
A:15:CYS:SG	A:28:CYS:SG	oxidized, CA 52.764, CB 39.487 ppm	oxidized, CA 55.158, CB 40.787 ppm	2.015

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-----
DCRYMFGDCEKDEDCCKHLGCKRKMKYCAWDFTFT
|||||||||||||||||||||||||||||||||||
DCRYMFGDCEKDEDCCKHLGCKRKMKYCAWDFTFT

Chain assignments

Entity_assembly_ID (NMR)	Auth_asym_ID (model)	Length	Unmapped	Conflict	Sequence coverage (%)
A	A	35	0	0	100.0

Content subtype: combined_30164_5t4r.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	OK	302		100.0 (chain: A, length: 35)
1	Distance restraints	CNS/XPLOR_distance_constraints_2	OK	649 (1)	noe	100.0 (chain: A, length: 35)
2	Distance restraints	CNS/XPLOR_distance_constraints_4	OK	38 (1)	hbond	57.1 (chain: A, length: 35)
1	Dihedral angle restraints	CNS/XPLOR_dihedral_3	Warning	62 (61)	.	100.0 (chain: A, length: 35)

Assigned chemical shifts

Saveframe: assigned_chemical_shifts_1
- Status: OK
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 35, Sequence coverage: 100.0%
- Total number of assignments
  - ¹H chemical shifts: 190
  - ¹³C chemical shifts: 79
  - ¹⁵N chemical shifts: 33
- Completeness of assignments
  - Entity_assemble_ID: A
- Redox state of CYS
- 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: 35, Sequence coverage: 100.0%
- Total number of restraints: 649
- Weight of restraints: 1.0 (649)
- Potential type of restraints: square-well-parabolic (649)
- Range of distance target values: 2.06, 19.5 (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
- Saveframe: CNS/XPLOR_distance_constraints_4
  - Status: OK
  - Experiment type: hbond
  - Sequence coverage of experimental data
    - Entity_assembly_ID: A, Chain length: 35, Sequence coverage: 57.1%
  - Total number of restraints: 38
  - Weight of restraints: 1.0 (38)
  - Potential type of restraints: square-well-parabolic (38)
  - Range of distance target values: 1.15, 1.65 (Å)
  - Histogram of distance target values
  - Distance restraints per residue
    - Chain_ID: A
  - Distance restraints on contact map

Dihedral angle restraints

Saveframe: CNS/XPLOR_dihedral_3
- Status: Warning
- Experiment type: .
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 35, Sequence coverage: 100.0%
- Total number of restraints: 61
- Total number of restraints per polymer type: 61
- Weight of restraints: 1.0 (61)
- Potential type of restraints: square-well-parabolic (61)
- Plot of Phi-Psi angle restraints
- Dihedral angle restraints per residue
  - Chain_ID: A

Keywords inhibitor cystine knot, tarantula peptide toxin, voltage sensor modifier, voltage-gated sodium channel modifier