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BMRB: 16202

2KGD

NMR Solution Structures of 3-oxo-butyl-ACP, an intermediate mimic from the actinorhodin polyketide synthase in Streptomyces coelicolor

Authors

Crump, M.P., Evans, S.E., Williams, C.

Assembly

3-oxo-butyl-ACP

Entity

1. 3-oxo-butyl-ACP (polymer, Thiol state: not present), 86 monomers, 9232.119 Da Detail

MATLLTTDDL RRALVESAGE TDGTDLSGDF LDLRFEDIGY DSLALMETAA RLESRYGVSI PDDVAGRVDT PRELLDLING ALAEAA

2. N~3~-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-N-{2-[(3-oxobutyl)sulfanyl]ethyl}-beta-alaninamide (non-polymer), 428.438 Da

Total weight

9660.557 Da

Max. entity weight

9232.119 Da

Source organism

Streptomyces coelicolor

Exptl. method

Refine. method

simulated annealing

Data set

assigned_chemical_shifts

Chem. Shift Complete

Sequence coverage: 98.8 %, Completeness: 97.4 %, Completeness (bb): 98.0 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	97.4 % (887 of 911)	96.8 % (447 of 462)	97.8 % (356 of 364)	98.8 % (84 of 85)
Backbone	98.0 % (502 of 512)	97.7 % (173 of 177)	98.0 % (246 of 251)	98.8 % (83 of 84)
Sidechain	96.9 % (463 of 478)	96.1 % (274 of 285)	97.9 % (188 of 192)	100.0 % (1 of 1)
Aromatic	100.0 % (36 of 36)	100.0 % (18 of 18)	100.0 % (18 of 18)
Methyl	98.3 % (118 of 120)	96.7 % (58 of 60)	100.0 % (60 of 60)

1. 3-oxo-butyl-ACP

MATLLTTDDL RRALVESAGE TDGTDLSGDF LDLRFEDIGY DSLALMETAA RLESRYGVSI PDDVAGRVDT PRELLDLING ALAEAA

Show sample condition

Sample

Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

LACS Plot; CA

Referencing offset: -0.29 ppm, Outliers: 1 Detail

LACS Plot; CB

Referencing offset: -0.29 ppm, Outliers: 1 Detail

LACS Plot; HA

Referencing offset: -0.12 ppm, Outliers: 1 Detail

LACS Plot; CO

Referencing offset: -0.04 ppm, Outliers: 3 Detail

Protein Blocks Logo

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

Release date

2009-04-21

Citation

Probing the Interactions of early polyketide intermediates with the Actinorhodin ACP from S. coelicolor A3(2)
Evans, S.E., Williams, C., Arthur, C.J., Ploskon, E., Wattana-Amorn, P., Cox, R.J., Crosby, J., Willis, C.L., Simpson, T.J., Crump, M.P.
J. Mol. Biol. (2009), 389, 511-528, PubMed 19361520 , DOI 10.1016/j.jmb.2009.03.072 , Abstract

Related entities 1. 3-oxo-butyl-ACP, : 1 : 10 : 3 : 64 entities Detail

Experiments performed 12 experiments Detail

1 2D 1H-15N HSQC

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

2 3D CBCA(CO)NH

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

3 3D C(CO)NH

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

4 3D HNCO

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

5 3D HNCACB

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

6 3D H(CCO)NH

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

7 3D HCCH-TOCSY

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

8 3D 1H-15N NOESY

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

9 3D 1H-13C NOESY

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

10 2D 13C,15N Filtered TOCSY

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

11 2D 13C,15N filtered NOESY

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

12 2D 13C F2 filtered NOESY

Instrument

Varian INOVA - 600 MHz 4 channel triple resonance probe equipped with XYZ gradients

Sample

State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 298 (±0.2) K, pH 5.5 (±0.2), Details Samples were 1mM NaN3, 5%/95% D2O/H2O in 600μL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The 3-oxo-butyl group and 4'PP chain were not isotopically labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	natural abundance		1.0 ~ 2.0 (±0.2) mM
2	potassium phosphate	natural abundance		20 (±2.0) mM
3	sodium azide	natural abundance		1 (±0.1) mM
4	H2O	natural abundance		95 %
5	D2O	natural abundance		5 %

NMR combined restraints 4 contents Detail

Properties

Input source #1: NMR data (NEF) - Assigned chemical shifts, Distance restraints, Dihedral angle restraints	combined_16202_2kgd.nef
Input source #2: Coordindates	2kgd.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	True (see coodinates for details)
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)

Ptnr_site_1	Ptnr_site_2	Redox_state_prediction_1	Redox_state_prediction_2	Distance (Å)
1:42:SER:OG	2:1:SXV:P24	unknown	unknown	n/a

Non-standard residues

Chain_ID	Seq_ID	Comp_ID	Chem_comp_name	Experimental evidences
B	1	SXV	N~3~-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-N-{2-[(3-oxobutyl)sulfanyl]ethyl}-beta-alaninamide	Assigned chemical shifts

Sequence alignments

Entity_assembly_ID (NMR): A vs Auth_asym_ID (model): A

--------10--------20--------30--------40--------50--------60--------70--------80------
MATLLTTDDLRRALVESAGETDGTDLSGDFLDLRFEDIGYDSLALMETAARLESRYGVSIPDDVAGRVDTPRELLDLINGALAEAA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MATLLTTDDLRRALVESAGETDGTDLSGDFLDLRFEDIGYDSLALMETAARLESRYGVSIPDDVAGRVDTPRELLDLINGALAEAA

Chain assignments

Entity_assembly_ID (NMR)	Auth_asym_ID (model)	Length	Unmapped	Conflict	Sequence coverage (%)
A	A	86	0	0	100.0

Content subtype: combined_16202_2kgd.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	933		98.8 (chain: A, length: 86), 100.0 (chain: B, length: 1)
1	Distance restraints	CNS/XPLOR_distance_constraints_2	OK	3628 (1)	noe	98.8 (chain: A, length: 86)
1	Dihedral angle restraints	CNS/XPLOR_dihedral_3	OK	120 (120)	.	83.7 (chain: A, length: 86)

Assigned chemical shifts

Saveframe: assigned_chem_shift_list_1
- Status: Warning
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 86, Sequence coverage: 98.8%
  - Entity_assembly_ID: B, Chain length: 1, Sequence coverage: 100.0%
- Total number of assignments
  - ¹H chemical shifts: 486
  - ¹³C chemical shifts: 355
  - ¹⁵N chemical shifts: 92
- Completeness of assignments
  - Entity_assemble_ID: A
  - Entity_assemble_ID: B
- Peptide bond of PRO
- 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: OK
- Experiment type: noe
- Sequence coverage of experimental data
  - Entity_assembly_ID: A, Chain length: 86, Sequence coverage: 98.8%
- Total number of restraints: 3627
- Weight of restraints: 1.0 (3627)
- Potential type of restraints: square-well-parabolic (3627)
- Range of distance target values: 1.7, 4.0 (Å)
- Histogram of distance target values
- Histogram of discrepancy in distance target values
- Distance restraints per residue
  - Chain_ID: A
  - Chain_ID: B
    None
- 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: 86, Sequence coverage: 83.7%
- Total number of restraints: 120
- Total number of restraints per polymer type: 120
- Weight of restraints: 1.0 (120)
- Potential type of restraints: square-well-parabolic (120)
- Plot of Phi-Psi angle restraints
- Dihedral angle restraints per residue
  - Chain_ID: A

Keywords ACP, Actinorhodin, Coelicolor, diketide, polyketide mimic