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BMRB: 25789

2N71

NMR structure of CmPI-II, a serin protease inhibitor isolated from mollusk Cenchitis muricatus

Authors

Cabrera-Munoz, A., Rojas, L., Alonso del Rivero Antigua, M., Pires, J.

Assembly

CmPI-II, a serin protease inhibitor

Entity

1. CmPI-II, a serin protease inhibitor (polymer, Thiol state: all disulfide bound), 50 monomers, 5486.007 Da Detail

AEDCVGRKAC TREWYPVCGS DGVTYSNPCN FSAQQEQCDP NITIAHMGEC

Formula weight

5486.007 Da

Entity Connection

disulfide 3 Detail

ID	Type	Value order	Atom ID 1	Atom ID 2
1	disulfide	sing	1:CYS4:SG	1:CYS38:SG
2	disulfide	sing	1:CYS10:SG	1:CYS29:SG
3	disulfide	sing	1:CYS18:SG	1:CYS50:SG

Source organism

Cenchritis muricatus

Exptl. method

Refine. method

simulated annealing, torsion angle dynamics

Data set

assigned_chemical_shifts

Chem. Shift Complete

Sequence coverage: 100.0 %, Completeness: 86.1 %, Completeness (bb): 95.2 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	86.1 % (459 of 533)	85.5 % (236 of 276)	83.7 % (170 of 203)	98.1 % (53 of 54)
Backbone	95.2 % (280 of 294)	98.0 % (99 of 101)	92.5 % (135 of 146)	97.9 % (46 of 47)
Sidechain	78.6 % (224 of 285)	78.3 % (137 of 175)	77.7 % (80 of 103)	100.0 % (7 of 7)
Aromatic	71.4 % (30 of 42)	71.4 % (15 of 21)	70.0 % (14 of 20)	100.0 % (1 of 1)
Methyl	94.1 % (32 of 34)	94.1 % (16 of 17)	94.1 % (16 of 17)

1. CmPI-II

AEDCVGRKAC TREWYPVCGS DGVTYSNPCN FSAQQEQCDP NITIAHMGEC

Show sample condition

Sample #1

Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
1	CmPI-II	natural abundance		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

Sample #2

Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
4	CmPI-II	[U-99% 15N]		1.2 mM
5	H2O	natural abundance		90 %
6	D2O	natural abundance		10 %

Sample #3

Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
7	CmPI-II	[U-99% 13C; U-99% 15N]		0.2 mM
8	H2O	natural abundance		90 %
9	D2O	natural abundance		10 %

LACS Plot; CA

Referencing offset: -0.32 ppm, Outliers: 1 Detail

LACS Plot; CB

Referencing offset: -0.32 ppm, Outliers: 1 Detail

LACS Plot; HA

Referencing offset: 0.07 ppm, Outliers: 2 Detail

LACS Plot; CO

Referencing offset: -0.09 ppm, Outliers: 1 Detail

Protein Blocks Logo

Calculated from 15 models in PDB: 2N71, Strand ID: A Detail

Release date

2016-06-30

Citation

1H, 13C and 15N resonance assignments and secondary structure analysis of CmPI-II, a serine protease inhibitor isolated from marine snail Cenchritis muricatus
Cabrera-Munoz, A., Rojas, L., Alonso del Rivero Antigua, M., Pires, J.
Biomol. NMR Assign. (2016), 10, 153-156, PubMed 26547437 , DOI 10.1007/s12104-015-9656-5 , Abstract

Related entities 1. CmPI-II, a serin protease inhibitor, : 1 : 1 : 265 entities Detail

Experiments performed 12 experiments Detail

1 2D 1H-1H TOCSY

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
1	CmPI-II	natural abundance		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

2 2D 1H-1H NOESY

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
1	CmPI-II	natural abundance		0.5 mM
2	H2O	natural abundance		90 %
3	D2O	natural abundance		10 %

3 2D 1H-15N HSQC

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
4	CmPI-II	[U-99% 15N]		1.2 mM
5	H2O	natural abundance		90 %
6	D2O	natural abundance		10 %

4 2D 1H-15N HSQC

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
7	CmPI-II	[U-99% 13C; U-99% 15N]		0.2 mM
8	H2O	natural abundance		90 %
9	D2O	natural abundance		10 %

5 2D 1H-13C HSQC

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
7	CmPI-II	[U-99% 13C; U-99% 15N]		0.2 mM
8	H2O	natural abundance		90 %
9	D2O	natural abundance		10 %

6 3D CBCA(CO)NH

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
7	CmPI-II	[U-99% 13C; U-99% 15N]		0.2 mM
8	H2O	natural abundance		90 %
9	D2O	natural abundance		10 %

7 3D 1H-15N NOESY

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
4	CmPI-II	[U-99% 15N]		1.2 mM
5	H2O	natural abundance		90 %
6	D2O	natural abundance		10 %

8 3D 1H-15N TOCSY

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
4	CmPI-II	[U-99% 15N]		1.2 mM
5	H2O	natural abundance		90 %
6	D2O	natural abundance		10 %

9 3D HNCO

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
7	CmPI-II	[U-99% 13C; U-99% 15N]		0.2 mM
8	H2O	natural abundance		90 %
9	D2O	natural abundance		10 %

10 3D HNCACB

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
7	CmPI-II	[U-99% 13C; U-99% 15N]		0.2 mM
8	H2O	natural abundance		90 %
9	D2O	natural abundance		10 %

11 3D HBHA(CO)NH

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
7	CmPI-II	[U-99% 13C; U-99% 15N]		0.2 mM
8	H2O	natural abundance		90 %
9	D2O	natural abundance		10 %

12 3D HCCH-TOCSY

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 K, pH 6.0

#	Name	Isotope labeling	Type	Concentration
7	CmPI-II	[U-99% 13C; U-99% 15N]		0.2 mM
8	H2O	natural abundance		90 %
9	D2O	natural abundance		10 %

NMR combined restraints 6 contents Detail

Properties

Input source #1: NMR data (NEF) - Assigned chemical shifts, Distance restraints, Dihedral angle restraints	combined_25789_2n71.nef
Input source #2: Coordindates	2n71.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:4:CYS:SG	A:38:CYS:SG	oxidized, CA 57.905, CB 38.22 ppm	oxidized, CA 54.813, CB 41.981 ppm	2.022
A:10:CYS:SG	A:29:CYS:SG	oxidized, CA 52.909, CB 41.08 ppm	oxidized, CA 60.24, CB 36.524 ppm	2.034
A:18:CYS:SG	A:50:CYS:SG	oxidized, CA 53.874, CB 38.042 ppm	oxidized, CA 56.403, CB 38.074 ppm	2.03

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
AEDCVGRKACTREWYPVCGSDGVTYSNPCNFSAQQEQCDPNITIAHMGEC
||||||||||||||||||||||||||||||||||||||||||||||||||
AEDCVGRKACTREWYPVCGSDGVTYSNPCNFSAQQEQCDPNITIAHMGEC

Chain assignments

Entity_assembly_ID (NMR)	Auth_asym_ID (model)	Length	Unmapped	Conflict	Sequence coverage (%)
A	A	50	0	0	100.0

Content subtype: combined_25789_2n71.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_chem_shift_list_1	Warning	461		100.0 (chain: A, length: 50)
1	Distance restraints	CNS/XPLOR_distance_constraints_2	Warning	697 (1)	noe	98.0 (chain: A, length: 50)
2	Distance restraints	CNS/XPLOR_distance_constraints_5	Warning	551 (1)	noe	100.0 (chain: A, length: 50)
3	Distance restraints	CNS/XPLOR_distance_constraints_3	OK	28 (1)	hbond	38.0 (chain: A, length: 50)
1	Dihedral angle restraints	CNS/XPLOR_dihedral_4	OK	72 (72)	.	98.0 (chain: A, length: 50)

Assigned chemical shifts

Saveframe: assigned_chem_shift_list_1
- Status: Warning
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 50, Sequence coverage: 100.0%
- Total number of assignments
  - ¹H chemical shifts: 236
  - ¹³C chemical shifts: 170
  - ¹⁵N chemical shifts: 55
- Completeness of assignments
  - Entity_assemble_ID: A
- Redox state of CYS
- Peptide bond of PRO
- Tautomeric state of HIS
- 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: 50, Sequence coverage: 98.0%
- Total number of restraints: 697
- Weight of restraints: 1.0 (697)
- Potential type of restraints: square-well-parabolic (697)
- Range of distance target values: 1.6, 4.0 (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_5
  - Status: Warning
  - Experiment type: noe
  - Sequence coverage of experimental data
    - Entity_assembly_ID: A, Chain length: 50, Sequence coverage: 100.0%
  - Total number of restraints: 550
  - Weight of restraints: 1.0 (550)
  - Potential type of restraints: square-well-parabolic (550)
  - Range of distance target values: 1.8, 4.0 (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_3
    - Status: OK
    - Experiment type: hbond
    - Sequence coverage of experimental data
      - Entity_assembly_ID: A, Chain length: 50, Sequence coverage: 38.0%
    - Total number of restraints: 28
    - Weight of restraints: 1.0 (28)
    - Potential type of restraints: square-well-parabolic (28)
    - Range of distance target values: 1.8, 2.8 (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_4
- Status: OK
- Experiment type: .
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 50, Sequence coverage: 98.0%
- Total number of restraints: 72
- Total number of restraints per polymer type: 72
- Weight of restraints: 1.0 (72)
- Potential type of restraints: square-well-parabolic (72)
- Plot of Phi-Psi angle restraints
- Dihedral angle restraints per residue
  - Chain_ID: A

Keywords Cenchritis muricatus, NMR solution structure, non-classical Kazal-inhibitor, protease inhibitor