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BMRB: 16200

2KGA

NMR Solution Structures of hexanoyl ACP (a non natural intermediate) from the actinorhodin polyketide synthase in Streptomyces coelicolor

Authors

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

Assembly

hexanoyl ACP

Entity

1. hexanoyl ACP (polymer, Thiol state: not present), 86 monomers, 9232.119 Da Detail

MATLLTTDDL RRALVESAGE TDGTDLSGDF LDLRFEDIGY DSLALMETAA RLESRYGVSI PDDVAGRVDT PRELLDLING ALAEAA

2. S-[2-({N-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl] hexanethioate (non-polymer), 456.491 Da

Total weight

9688.61 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: 96.5 %, Completeness: 96.4 %, Completeness (bb): 96.1 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	96.4 % (878 of 911)	96.8 % (447 of 462)	95.9 % (349 of 364)	96.5 % (82 of 85)
Backbone	96.1 % (492 of 512)	96.6 % (171 of 177)	95.6 % (240 of 251)	96.4 % (81 of 84)
Sidechain	96.7 % (462 of 478)	96.8 % (276 of 285)	96.4 % (185 of 192)	100.0 % (1 of 1)
Aromatic	100.0 % (36 of 36)	100.0 % (18 of 18)	100.0 % (18 of 18)
Methyl	96.7 % (116 of 120)	96.7 % (58 of 60)	96.7 % (58 of 60)

1. hexanoyl 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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: -0.32 ppm, Outliers: 1 Detail

LACS Plot; CB

Referencing offset: -0.32 ppm, Outliers: 1 Detail

LACS Plot; HA

Referencing offset: -0.11 ppm, Outliers: 1 Detail

LACS Plot; CO

Referencing offset: 0.01 ppm, Outliers: 2 Detail

Protein Blocks Logo

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

Release date

2009-04-16

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. hexanoyl ACP, : 1 : 10 : 3 : 64 entities Detail

Experiments performed 12 experiments Detail

1 2D 1H-15N HSQC

Instrument

Varian INOVA - 600 MHz 4-channel INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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 INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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 INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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 INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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 INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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 INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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 1H-15N NOESY

Instrument

Varian INOVA - 600 MHz 4-channel INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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-13C NOESY

Instrument

Varian INOVA - 600 MHz 4-channel INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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 2D 13C,15N Filtered NOESY

Instrument

Varian INOVA - 600 MHz 4-channel INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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 INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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 F2 13C Filtered NOESY

Instrument

Varian INOVA - 600 MHz 4-channel INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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 3D HCCH-TOCSY

Instrument

Varian INOVA - 600 MHz 4-channel INOVA 600 MHz equipped with a triple resonance coil and triple axis 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 600uL potassium phosphate buffer (20mM, pH 5.5) and 1-2mM derivatised ACP. The derivatised sidechain is not uniformly isotope labelled.

#	Name	Isotope labeling	Type	Concentration
1	ACP	[U-98% 13C; U-98% 15N]		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_16200_2kga.nef
Input source #2: Coordindates	2kga.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:SXH:P24	unknown	unknown	n/a

Non-standard residues

Chain_ID	Seq_ID	Comp_ID	Chem_comp_name	Experimental evidences
B	1	SXH	S-[2-({N-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl] hexanethioate	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_16200_2kga.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	928		96.5 (chain: A, length: 86), 100.0 (chain: B, length: 1)
1	Distance restraints	CNS/XPLOR_distance_constraints_2	OK	3664 (1)	noe	98.8 (chain: A, length: 86)
1	Dihedral angle restraints	CNS/XPLOR_dihedral_3	OK	128 (128)	.	87.2 (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: 96.5%

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

Entity_assembly_ID: B, Chain length: 1, Sequence coverage: 100.0%

-
X
|
X

Total number of assignments
- ¹H chemical shifts: 482
- ¹³C chemical shifts: 355
- ¹⁵N chemical shifts: 91

Completeness of assignments

Entity_assemble_ID: A

Excluded Atom_ID in statistics

Comp_index_ID	Comp_ID	Atom_ID	CS value (ppm)
11	ARG	CZ	159.547
12	ARG	CZ	159.628
34	ARG	CZ	159.886
51	ARG	CZ	159.644
55	ARG	CZ	159.95
67	ARG	CZ	159.736
72	ARG	HH11	6.892
72	ARG	HH12	6.892
72	ARG	HH21	6.892
72	ARG	HH22	6.892
72	ARG	CZ	159.429
72	ARG	NH1	71.196
72	ARG	NH2	71.196

All atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	462	447	96.8
¹³C chemical shifts	364	348	95.6
¹⁵N chemical shifts	92	89	96.7

Backbone atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	177	171	96.6
¹³C chemical shifts	172	163	94.8
¹⁵N chemical shifts	84	81	96.4

Side chain atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	285	276	96.8
¹³C chemical shifts	192	185	96.4
¹⁵N chemical shifts	8	8	100.0

Methyl group atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	62	58	93.5
¹³C chemical shifts	62	58	93.5

Aromatic group atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	18	18	100.0
¹³C chemical shifts	18	18	100.0

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: 3664
- Weight of restraints: 1.0 (3664)
- Potential type of restraints: square-well-parabolic (3664)
- Range of distance target values: 1.65, 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: 87.2%
- Total number of restraints: 128
- Total number of restraints per polymer type: 128
- Weight of restraints: 1.0 (128)
- Potential type of restraints: square-well-parabolic (128)
- Plot of Phi-Psi angle restraints
- Dihedral angle restraints per residue
  - Chain_ID: A

Keywords ACP, Conformational Change, Fatty Acid, Hexanoyl, Polyketide