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Found 1 Document (1.046 seconds)

BMRB: 15966

2K73

Solution NMR structure of integral membrane protein DsbB

Authors

Zhou, Y., Cierpicki, T., Flores Jimenez, R.H., Lukasik, S.M., Ellena, J.F., Cafiso, D.S., Kadukura, H., Beckwith, J., Bushweller, J.H.

Assembly

DsbB

Entity

1. DsbB (polymer, Thiol state: all disulfide bound), 183 monomers, 20925.58 Da Detail

MLRFLNQASQ GRGAWLLMAF TALALELTAL WFQHVMLLKP CVLSIYERAA LFGVLGAALI GAIAPKTPLR YVAMVIWLYS AFRGVQLTYE HTMLQLYPSP FATSDFMVRF PEWLPLDKWV PQVFVASGDC AERQWDFLGL EMPQWLLGIF IAYLIVAVLV VISQPFKAKK RDLFGRGHHH HHH

Formula weight

20925.58 Da

Entity Connection

disulfide 1 Detail

ID	Type	Value order	Atom ID 1	Atom ID 2
1	disulfide	sing	1:CYS41:SG	1:CYS130:SG

Source organism

Escherichia coli

Exptl. method

solution NMR

Refine. method

simulated annealing

Data set

assigned_chemical_shifts

Chem. Shift Complete

Sequence coverage: 96.7 %, Completeness: 40.3 %, Completeness (bb): 79.4 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	40.3 % (905 of 2247)	18.9 % (218 of 1152)	56.7 % (513 of 905)	91.6 % (174 of 190)
Backbone	79.4 % (856 of 1078)	49.9 % (183 of 367)	94.1 % (506 of 538)	96.5 % (167 of 173)
Sidechain	15.7 % (210 of 1341)	4.5 % (35 of 785)	31.2 % (168 of 539)	41.2 % (7 of 17)
Aromatic	4.8 % (14 of 294)	4.8 % (7 of 147)	0.0 % (0 of 140)	100.0 % (7 of 7)
Methyl	14.7 % (37 of 252)	8.7 % (11 of 126)	20.6 % (26 of 126)

1. Disulfide bond formation protein B

Show sample condition

Sample #1

Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
1	DsbB[CSSC]	[U-13C; U-15N; U-2H]	1.2 mM
2	DPC	[U-2H]	100 mM
3	sodium phosphate	natural abundance	25 mM
4	potassium chloride	natural abundance	50 mM

Sample #2

Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
5	DsbB[CSSC]	I,L,V methyl protonated, [U-13C; U-15N; U-2H]	1.5 mM
6	DPC	[U-2H]	100 mM
7	sodium phosphate	natural abundance	25 mM
8	potassium chloride	natural abundance	50 mM

Sample #3

Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2, Details single Cys mutants to incorporate MTSL/dMTSL for PRE measurements

#	Name	Isotope labeling	Concentration
9	DsbB[CSSC] single Cys mutants	[U-15N; U-2H]	0.5 ~ 1.0 mM
10	DPC	natural abundance	100 mM
11	sodium phosphate	natural abundance	25 mM
12	potassium chloride	natural abundance	50 mM

LACS Plot; CA

Atom type	Mol. common name	Atom group	Ref. method	Ref. type	Shift ratio
¹³C	DSS	methyl protons	external	indirect	0.2514495
¹H	DSS	methyl protons	external	direct	1.0
¹⁵N	DSS	methyl protons	external	indirect	0.1013291

Referencing offset: 0.46 ppm, Outliers: 1 Detail

LACS Plot; CB

Atom type	Mol. common name	Atom group	Ref. method	Ref. type	Shift ratio
¹³C	DSS	methyl protons	external	indirect	0.2514495
¹H	DSS	methyl protons	external	direct	1.0
¹⁵N	DSS	methyl protons	external	indirect	0.1013291

Referencing offset: 0.46 ppm, Outliers: 1 Detail

LACS Plot; CO

Atom type	Mol. common name	Atom group	Ref. method	Ref. type	Shift ratio
¹³C	DSS	methyl protons	external	indirect	0.2514495
¹H	DSS	methyl protons	external	direct	1.0
¹⁵N	DSS	methyl protons	external	indirect	0.1013291

Referencing offset: 0.25 ppm, Outliers: 3 Detail

Protein Blocks Logo

Calculated from 20 models in PDB: 2K73, Strand ID: A Detail

Release date

2008-12-11

Citation

NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation
Zhou, Y., Cierpicki, T., Flores Jimenez, R.H., Lukasik, S.M., Ellena, J.F., Cafiso, D.S., Kadukura, H., Beckwith, J., Bushweller, J.H.
Mol. Cell (2008), 31, 896-908, PubMed 18922471 , DOI 10.1016/j.molcel.2008.08.028 , Abstract

Related entities 1. DsbB, : 1 : 1 : 8 : 53 entities Detail

Interaction partners 1. DsbB, : 1 interactors Detail

More details for 1 interactors identified by 1 citations

Experiments performed 13 experiments Detail

1 3D HNCO

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
1	DsbB[CSSC]	[U-13C; U-15N; U-2H]	1.2 mM
2	DPC	[U-2H]	100 mM
3	sodium phosphate	natural abundance	25 mM
4	potassium chloride	natural abundance	50 mM

2 3D HN(CA)CO

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
1	DsbB[CSSC]	[U-13C; U-15N; U-2H]	1.2 mM
2	DPC	[U-2H]	100 mM
3	sodium phosphate	natural abundance	25 mM
4	potassium chloride	natural abundance	50 mM

3 3D HNCA

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
1	DsbB[CSSC]	[U-13C; U-15N; U-2H]	1.2 mM
2	DPC	[U-2H]	100 mM
3	sodium phosphate	natural abundance	25 mM
4	potassium chloride	natural abundance	50 mM

4 3D HN(CO)CA

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
1	DsbB[CSSC]	[U-13C; U-15N; U-2H]	1.2 mM
2	DPC	[U-2H]	100 mM
3	sodium phosphate	natural abundance	25 mM
4	potassium chloride	natural abundance	50 mM

5 3D HNCACB

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
1	DsbB[CSSC]	[U-13C; U-15N; U-2H]	1.2 mM
2	DPC	[U-2H]	100 mM
3	sodium phosphate	natural abundance	25 mM
4	potassium chloride	natural abundance	50 mM

6 2D 1H-15N HSQC

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
1	DsbB[CSSC]	[U-13C; U-15N; U-2H]	1.2 mM
2	DPC	[U-2H]	100 mM
3	sodium phosphate	natural abundance	25 mM
4	potassium chloride	natural abundance	50 mM

7 3D HNCO based RDC experiments

Instrument

Varian INOVA - 600 MHz

Sample

State anisotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
1	DsbB[CSSC]	[U-13C; U-15N; U-2H]	1.2 mM
2	DPC	[U-2H]	100 mM
3	sodium phosphate	natural abundance	25 mM
4	potassium chloride	natural abundance	50 mM

8 3D 1H-15N NOESY

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
5	DsbB[CSSC]	I,L,V methyl protonated, [U-13C; U-15N; U-2H]	1.5 mM
6	DPC	[U-2H]	100 mM
7	sodium phosphate	natural abundance	25 mM
8	potassium chloride	natural abundance	50 mM

9 3D 1H-13C NOESY

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
5	DsbB[CSSC]	I,L,V methyl protonated, [U-13C; U-15N; U-2H]	1.5 mM
6	DPC	[U-2H]	100 mM
7	sodium phosphate	natural abundance	25 mM
8	potassium chloride	natural abundance	50 mM

10 2D 1H-13C HSQC

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
5	DsbB[CSSC]	I,L,V methyl protonated, [U-13C; U-15N; U-2H]	1.5 mM
6	DPC	[U-2H]	100 mM
7	sodium phosphate	natural abundance	25 mM
8	potassium chloride	natural abundance	50 mM

11 3D 13C-13C NOESY

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
5	DsbB[CSSC]	I,L,V methyl protonated, [U-13C; U-15N; U-2H]	1.5 mM
6	DPC	[U-2H]	100 mM
7	sodium phosphate	natural abundance	25 mM
8	potassium chloride	natural abundance	50 mM

12 3D HMCM[CG]CBCA

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2

#	Name	Isotope labeling	Concentration
5	DsbB[CSSC]	I,L,V methyl protonated, [U-13C; U-15N; U-2H]	1.5 mM
6	DPC	[U-2H]	100 mM
7	sodium phosphate	natural abundance	25 mM
8	potassium chloride	natural abundance	50 mM

13 2D 1H-15N HSQC

Instrument

Varian INOVA - 600 MHz

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 313 K, pH 6.2, Details single Cys mutants to incorporate MTSL/dMTSL for PRE measurements

#	Name	Isotope labeling	Concentration
9	DsbB[CSSC] single Cys mutants	[U-15N; U-2H]	0.5 ~ 1.0 mM
10	DPC	natural abundance	100 mM
11	sodium phosphate	natural abundance	25 mM
12	potassium chloride	natural abundance	50 mM

NMR combined restraints 6 contents Detail

Properties

Input source #1: NMR data (NEF) - Assigned chemical shifts, Distance restraints, Dihedral angle restraints, RDC restraints	combined_15966_2k73.nef
Input source #2: Coordindates	2k73.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:41:CYS:SG	A:130:CYS:SG	oxidized, CA 56.18, CB 50.684 ppm	oxidized, CA 54.992, CB 38.294 ppm	2.02

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--------60--------70--------80--------90-------100
MLRFLNQASQGRGAWLLMAFTALALELTALWFQHVMLLKPCVLSIYERAALFGVLGAALIGAIAPKTPLRYVAMVIWLYSAFRGVQLTYEHTMLQLYPSP
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MLRFLNQASQGRGAWLLMAFTALALELTALWFQHVMLLKPCVLSIYERAALFGVLGAALIGAIAPKTPLRYVAMVIWLYSAFRGVQLTYEHTMLQLYPSP

-------110-------120-------130-------140-------150-------160-------170-------180---
FATSDFMVRFPEWLPLDKWVPQVFVASGDCAERQWDFLGLEMPQWLLGIFIAYLIVAVLVVISQPFKAKKRDLFGRGHHHHHH
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
FATSDFMVRFPEWLPLDKWVPQVFVASGDCAERQWDFLGLEMPQWLLGIFIAYLIVAVLVVISQPFKAKKRDLFGRGHHHHHH

Chain assignments

Entity_assembly_ID (NMR)	Auth_asym_ID (model)	Length	Unmapped	Conflict	Sequence coverage (%)
A	A	183	0	0	100.0

Content subtype: combined_15966_2k73.nef

#	Content subtype	Saveframe	Status	# of rows (sets)	Experiment type	Sequence coverage (%)
1	Covalent bonds	assembly	OK	1		No information
1	Assigned chemical shifts	assigned_chem_shift_list_1	Warning	839		95.1 (chain: A, length: 183)
1	Distance restraints	CNS/XPLOR_distance_constraints_3	OK	608 (1)	noe	80.9 (chain: A, length: 183)
2	Distance restraints	CNS/XPLOR_distance_constraints_9	OK	107 (1)	hbond	65.0 (chain: A, length: 183)
1	Dihedral angle restraints	CNS/XPLOR_dihedral_7	OK	295 (295)	.	84.7 (chain: A, length: 183)
1	RDC restraints	CNS/XPLOR_dipolar_coupling_5	OK	335 (335)	.	77.6 (chain: A, length: 183)

Assigned chemical shifts

Saveframe: assigned_chem_shift_list_1
- Status: Warning
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 183, Sequence coverage: 95.1%
- Total number of assignments
  - ¹H chemical shifts: 170
  - ¹³C chemical shifts: 499
  - ¹⁵N chemical shifts: 170
- Completeness of assignments
  - Entity_assemble_ID: A
- Redox state of CYS
- Peptide bond of PRO
- Histogram of normalized assigned chemical shifts
- Random coil index and derived parameters (S² and NMR RMSD)
  - Chain_ID: A

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	1152	169	14.7
¹³C chemical shifts	905	499	55.1
¹⁵N chemical shifts	199	170	85.4

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	367	162	44.1
¹³C chemical shifts	366	341	93.2
¹⁵N chemical shifts	173	163	94.2

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	785	7	0.9
¹³C chemical shifts	539	158	29.3
¹⁵N chemical shifts	26	7	26.9

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	133	0	0.0
¹³C chemical shifts	133	20	15.0

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	147	7	4.8
¹³C chemical shifts	140	0	0.0
¹⁵N chemical shifts	7	7	100.0

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: 183, Sequence coverage: 80.9%
- Total number of restraints: 607
- Weight of restraints: 1.0 (607)
- Potential type of restraints: square-well-parabolic (607)
- Range of distance target values: 2.15, 4.15 (Å)
- Histogram of distance target values
- Distance restraints per residue
  - Chain_ID: A
- Distance restraints on contact map
- Saveframe: CNS/XPLOR_distance_constraints_9
  - Status: OK
  - Experiment type: hbond
  - Sequence coverage of experimental data
    - Entity_assembly_ID: A, Chain length: 183, Sequence coverage: 65.0%
  - Total number of restraints: 107
  - Weight of restraints: 1.0 (107)
  - Potential type of restraints: square-well-parabolic (107)
  - Range of distance target values: 2.05, 2.1 (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_7
- Status: OK
- Experiment type: .
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 183, Sequence coverage: 84.7%
- Total number of restraints: 295
- Total number of restraints per polymer type: 295
- Weight of restraints: 1.0 (295)
- Potential type of restraints: square-well-parabolic (295)
- Plot of Phi-Psi angle restraints
- Dihedral angle restraints per residue
  - Chain_ID: A

RDC restraints

Saveframe: CNS/XPLOR_dipolar_coupling_5

Status: OK
Experiment type: .

Sequence coverage of experimental data

Entity_assembly_ID: A, Chain length: 183, Sequence coverage: 77.6%

--------10--------20--------30--------40--------50--------60--------70--------80--------90-------100
MLRFLNQASQGRGAWLLMAFTALALELTALWFQHVMLLKPCVLSIYERAALFGVLGAALIGAIAPKTPLRYVAMVIWLYSAFRGVQLTYEHTMLQLYPSP
  ||||||||||||||||||  |||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||  
..RFLNQASQGRGAWLLMAF..LALELTALWFQH.MLLKPCVLSIYERAALFGVLGAALIGAIAPKTPLRYVAMVIWLYSAFRGVQLTYEHTMLQLYP..
--------10--------20--------30--------40--------50--------60--------70--------80--------90-------100

-------110-------120-------130-------140-------150-------160-------170-------180---
FATSDFMVRFPEWLPLDKWVPQVFVASGDCAERQWDFLGLEMPQWLLGIFIAYLIVAVLVVISQPFKAKKRDLFGRGHHHHHH
            ||||||||||||||||||||||||||||||||||||||||| ||||||||                     
............WLPLDKWVPQVFVASGDCAERQWDFLGLEMPQWLLGIFIAY.IVAVLVVI                     
-------110-------120-------130-------140-------150-------160--

Total number of restraints: 335
Potential type of restraints: undefined (335)
Range of observed RDC values: 100.0, -100.0 (Hz)
RDC restraints per residue
- Chain_ID: A
  None

Keywords disulfide bond, DsbB, membrane protein, redox enzyme

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Found 1 Document (1.046 seconds)

BMRB: 15966

Sample #1

Sample #2

Sample #3

Chemical shift reference

Chemical shift reference

Chemical shift reference

Related entities 1. DsbB, : 1 : 1 : 8 : 53 entities Detail

Interaction partners 1. DsbB, : 1 interactors Detail

Experiments performed 13 experiments Detail

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NMR combined restraints 6 contents Detail