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BMRB: 16428

2KMG

The structure of the KlcA and ArdB proteins show a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro.

Authors

Serfiotis-Mitsa, D., Herbert, A.P., Roberts, G.A., Soares, D.C., White, J.H., Blakely, G.W., Uhrin, D., Dryden, D.T.F.

Assembly

KlcA and ArdB proteins

Entity

1. KlcA and ArdB proteins (polymer, Thiol state: all free), 142 monomers, 15659.50 Da Detail

MNTEEQPVTA SLVAEAQRLD FLPTYFGPRL MMRGEALVYA WMRRLCERYN GAYWHYYALS DGGFYMAPDL AGRLEIEVNG NGFRGELSAD AAGIVATLFA LGQLAAEIAD TDAADALIDR YHFLRGFAAG HPEAAAIYRA ID

Formula weight

15659.5 Da

Source organism

Bordetella pertussis

Exptl. method

solution NMR

Refine. method

torsion angle dynamics, simulated annealing

Data set

assigned_chemical_shifts, RDCs

Chem. Shift Complete

Sequence coverage: 98.6 %, Completeness: 90.1 %, Completeness (bb): 97.0 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	90.1 % (1432 of 1590)	90.4 % (733 of 811)	88.2 % (558 of 633)	96.6 % (141 of 146)
Backbone	97.0 % (817 of 842)	96.2 % (281 of 292)	97.8 % (404 of 413)	96.4 % (132 of 137)
Sidechain	84.6 % (742 of 877)	87.1 % (452 of 519)	80.5 % (281 of 349)	100.0 % (9 of 9)
Aromatic	44.4 % (79 of 178)	53.9 % (48 of 89)	33.3 % (29 of 87)	100.0 % (2 of 2)
Methyl	97.6 % (162 of 166)	97.6 % (81 of 83)	97.6 % (81 of 83)

1. KlcA and ArdB proteins

MNTEEQPVTA SLVAEAQRLD FLPTYFGPRL MMRGEALVYA WMRRLCERYN GAYWHYYALS DGGFYMAPDL AGRLEIEVNG NGFRGELSAD AAGIVATLFA LGQLAAEIAD TDAADALIDR YHFLRGFAAG HPEAAAIYRA ID

Show sample condition

Sample #1

Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

Sample #2

Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
6	KlcA	[U-98% 15N]	0.7 mM
7	sodium acetate	[U-100% 2H]	20 mM
8	sodium azide	natural abundance	0.05 %
9	Pf1 phage	natural abundance	6.25 mg
10	H2O	natural abundance	90 %
11	D2O	natural abundance	10 %

Sample #3

Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
12	KlcA	[U-98% 15N]	0.7 mM
13	sodium acetate	[U-100% 2H]	20 mM
14	sodium azide	natural abundance	0.05 %
15	H2O	natural abundance	90 %
16	D2O	natural abundance	10 %

LACS Plot; CA

Atom type	Mol. common name	Atom group	Value	Ref. method	Ref. type	Shift ratio
¹³C	DSS	methyl protons	0.0 ppm	na	indirect	0.2514495
¹H	water	protons	4.768 ppm	internal	direct	1.0
¹⁵N	DSS	methyl protons	0.0 ppm	na	indirect	0.1013291

Referencing offset: 0.0 ppm, Outliers: 2 Detail

LACS Plot; CB

Atom type	Mol. common name	Atom group	Value	Ref. method	Ref. type	Shift ratio
¹³C	DSS	methyl protons	0.0 ppm	na	indirect	0.2514495
¹H	water	protons	4.768 ppm	internal	direct	1.0
¹⁵N	DSS	methyl protons	0.0 ppm	na	indirect	0.1013291

Referencing offset: 0.0 ppm, Outliers: 2 Detail

LACS Plot; HA

Atom type	Mol. common name	Atom group	Value	Ref. method	Ref. type	Shift ratio
¹³C	DSS	methyl protons	0.0 ppm	na	indirect	0.2514495
¹H	water	protons	4.768 ppm	internal	direct	1.0
¹⁵N	DSS	methyl protons	0.0 ppm	na	indirect	0.1013291

Referencing offset: -0.02 ppm, Outliers: 1 Detail

LACS Plot; CO

Atom type	Mol. common name	Atom group	Value	Ref. method	Ref. type	Shift ratio
¹³C	DSS	methyl protons	0.0 ppm	na	indirect	0.2514495
¹H	water	protons	4.768 ppm	internal	direct	1.0
¹⁵N	DSS	methyl protons	0.0 ppm	na	indirect	0.1013291

Referencing offset: -0.19 ppm, Outliers: 2 Detail

Protein Blocks Logo

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

RDC

110 RDC values in 1 lists
Field strength (¹H) 800.014 MHz, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2) Detail

Release date

2010-02-01

Citation

The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro
Serfiotis-Mitsa, D., Herbert, A.P., Roberts, G.A., Soares, D.C., White, J.H., Blakely, G.W., Uhrin, D., Dryden, D.T.F.
Nucleic Acids Res. (2010), 38, 1723-1737, PubMed 20007596 , DOI 10.1093/nar/gkp1144 , Abstract

Related entities 1. KlcA and ArdB proteins, : 1 : 11 entities Detail

Experiments performed 21 experiments Detail

1 2D 1H-15N HSQC

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

2 3D CBCA(CO)NH

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

3 3D CBCANH

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

4 3D HNCO

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

5 3D HNCACO

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

6 3D HBHANH

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

7 3D HBHA(CO)NH

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

8 2D 1H-13C HSQC (Aromatic Selective)

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

9 3D H(CCO)NH

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

10 3D C(CCO)NH

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

11 3D HCCH-TOCSY

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

12 2D 1H-13C HSQC

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

13 3D 1H-15N NOESY

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

14 3D 1H-13C NOESY

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

15 2D HBCBCGCDHD

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

16 2D HBCBCGCDCEHE

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

17 T1

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
12	KlcA	[U-98% 15N]	0.7 mM
13	sodium acetate	[U-100% 2H]	20 mM
14	sodium azide	natural abundance	0.05 %
15	H2O	natural abundance	90 %
16	D2O	natural abundance	10 %

18 T2

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
12	KlcA	[U-98% 15N]	0.7 mM
13	sodium acetate	[U-100% 2H]	20 mM
14	sodium azide	natural abundance	0.05 %
15	H2O	natural abundance	90 %
16	D2O	natural abundance	10 %

19 Heteronuclear NOE

Instrument

Bruker Avance - 600 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
12	KlcA	[U-98% 15N]	0.7 mM
13	sodium acetate	[U-100% 2H]	20 mM
14	sodium azide	natural abundance	0.05 %
15	H2O	natural abundance	90 %
16	D2O	natural abundance	10 %

20 IPAP for RDC measurement

Instrument

Bruker Avance - 600 MHz

Sample

State anisotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
6	KlcA	[U-98% 15N]	0.7 mM
7	sodium acetate	[U-100% 2H]	20 mM
8	sodium azide	natural abundance	0.05 %
9	Pf1 phage	natural abundance	6.25 mg
10	H2O	natural abundance	90 %
11	D2O	natural abundance	10 %

21 2D 1H-13C HSQC

Instrument

Bruker Avance - 800 MHz

Sample

State isotropic, Solvent system 90% H2O/10% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 6.0 (±0.2)

#	Name	Isotope labeling	Concentration
1	KlcA	[U-98% 13C; U-98% 15N]	0.7 mM
2	sodium acetate	[U-100% 2H]	20 mM
3	sodium azide	natural abundance	0.05 %
4	H2O	natural abundance	90 %
5	D2O	natural abundance	10 %

NMR combined restraints 4 contents Detail

Properties

Input source #1: NMR data (NEF) - Assigned chemical shifts, Distance restraints, Dihedral angle restraints	combined_16428_2kmg.nef
Input source #2: Coordindates	2kmg.cif
Diamagnetism of the molecular assembly	True (excluding Oxygen atoms)
Whether the assembly has a disulfide bond	None
Whether the assembly has a other bond	None
Whether the assembly contains a cyclic polymer	None
Overall data processing status	Warning

Disulfide bonds

None

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
MNTEEQPVTASLVAEAQRLDFLPTYFGPRLMMRGEALVYAWMRRLCERYNGAYWHYYALSDGGFYMAPDLAGRLEIEVNGNGFRGELSADAAGIVATLFA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MNTEEQPVTASLVAEAQRLDFLPTYFGPRLMMRGEALVYAWMRRLCERYNGAYWHYYALSDGGFYMAPDLAGRLEIEVNGNGFRGELSADAAGIVATLFA

-------110-------120-------130-------140--
LGQLAAEIADTDAADALIDRYHFLRGFAAGHPEAAAIYRAID
||||||||||||||||||||||||||||||||||||||||||
LGQLAAEIADTDAADALIDRYHFLRGFAAGHPEAAAIYRAID

Chain assignments

Entity_assembly_ID (NMR)	Auth_asym_ID (model)	Length	Unmapped	Conflict	Sequence coverage (%)
A	A	142	0	0	100.0

Content subtype: combined_16428_2kmg.nef

#	Content subtype	Saveframe	Status	# of rows (sets)	Experiment type	Sequence coverage (%)
1	Assigned chemical shifts	assigned_chem_shift_list_1	Warning	1439		97.9 (chain: A, length: 142)
1	Distance restraints	CNS/XPLOR_distance_constraints_2	Warning	3058 (1)	noe	97.2 (chain: A, length: 142)
1	Dihedral angle restraints	CNS/XPLOR_dihedral_3	OK	228 (228)	.	95.1 (chain: A, length: 142)

Assigned chemical shifts

Saveframe: assigned_chem_shift_list_1
- Status: Warning
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 142, Sequence coverage: 97.9%
- Total number of assignments
  - ¹H chemical shifts: 731
  - ¹³C chemical shifts: 559
  - ¹⁵N chemical shifts: 149
- 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

Comp_index_ID	Comp_ID	Atom_ID	CS value (ppm)
2	ASN	CG	176.894
6	GLN	CD	180.63
17	GLN	CD	180.621
50	ASN	CG	177.677
79	ASN	CG	176.238

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	811	731	90.1
¹³C chemical shifts	633	554	87.5
¹⁵N chemical shifts	157	149	94.9

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	292	283	96.9
¹³C chemical shifts	284	277	97.5
¹⁵N chemical shifts	137	131	95.6

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	519	448	86.3
¹³C chemical shifts	349	277	79.4
¹⁵N chemical shifts	20	18	90.0

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	88	83	94.3
¹³C chemical shifts	88	83	94.3

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	89	40	44.9
¹³C chemical shifts	87	26	29.9
¹⁵N chemical shifts	2	2	100.0

Distance restraints

Saveframe: CNS/XPLOR_distance_constraints_2
- Status: Warning
- Experiment type: noe
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 142, Sequence coverage: 97.2%
- Total number of restraints: 3051
- Weight of restraints: 1.0 (3051)
- Potential type of restraints: square-well-parabolic (3051)
- Range of distance target values: 2.17, 3.65 (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: 142, Sequence coverage: 95.1%
- Total number of restraints: 228
- Total number of restraints per polymer type: 228
- Weight of restraints: 1.0 (228)
- Potential type of restraints: square-well-parabolic (228)
- Plot of Phi-Psi angle restraints
- Dihedral angle restraints per residue
  - Chain_ID: A

Keywords Anti-restriction, ArdB, KlcA, NMR spectroscopy

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Found 1 Document (0.807 seconds)

BMRB: 16428

Sample #1

Sample #2

Sample #3

Chemical shift reference

Chemical shift reference

Chemical shift reference

Chemical shift reference

Related entities 1. KlcA and ArdB proteins, : 1 : 11 entities Detail

Experiments performed 21 experiments Detail

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NMR combined restraints 4 contents Detail