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BMRB: 16201

2KGC

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

Authors

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

Assembly

octanoyl ACP

Entity

1. octanoyl 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] octanethioate (non-polymer), 484.544 Da

Total weight

9716.663 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: 97.3 %, Completeness (bb): 97.9 % Detail

Polymer type: polypeptide(L)

	Total	¹H	¹³C	¹⁵N
All	97.3 % (886 of 911)	96.8 % (447 of 462)	97.5 % (355 of 364)	98.8 % (84 of 85)
Backbone	97.9 % (501 of 512)	97.7 % (173 of 177)	97.6 % (245 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. octanoyl 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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

LACS Plot; CA

Referencing offset: -0.43 ppm, Outliers: 1 Detail

LACS Plot; CB

Referencing offset: -0.43 ppm, Outliers: 1 Detail

LACS Plot; HA

Referencing offset: -0.11 ppm, Outliers: 1 Detail

LACS Plot; CO

Referencing offset: -0.03 ppm, Outliers: 2 Detail

Protein Blocks Logo

Calculated from 20 models in PDB: 2KGC, 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. octanoyl ACP, : 1 : 10 : 3 : 64 entities Detail

Experiments performed 13 experiments Detail

1 2D 1H-15N HSQC

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

2 3D CBCA(CO)NH

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

3 3D C(CO)NH

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

4 3D HNCO

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

5 3D HNCACB

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

6 3D H(CCO)NH

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

7 3D HCCH-TOCSY

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

8 3D HNHA

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

9 3D 1H-15N NOESY

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

10 3D 1H-13C NOESY

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

11 2D 13C,15N filtered NOESY

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

12 2D 13C,15N filtered TOCSY

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

13 2D F2-13C filtered NOESY

Instrument

Varian INOVA - 600 MHz INOVA 600 eqipped with a RT triple resonance probe with XYZ triple axis gradients and 4 channels.

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

#	Name	Isotope labeling	Type	Concentration
1	potassium phosphate	natural abundance		20 (±2.0) mM
2	sodium azide	natural abundance		1 (±0.1) mM
3	4 -phosphopantetheine chain acylated with an octanoyl group	natural abundance		1.0 ~ 2.0 (±0.2) mM

NMR combined restraints 4 contents Detail

Properties

Input source #1: NMR data (NEF) - Assigned chemical shifts, Distance restraints, Dihedral angle restraints	combined_16201_2kgc.nef
Input source #2: Coordindates	2kgc.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:SXO:P24	unknown	unknown	n/a

Non-standard residues

Chain_ID	Seq_ID	Comp_ID	Chem_comp_name	Experimental evidences
B	1	SXO	S-[2-({N-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl] octanethioate	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_16201_2kgc.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	946		98.8 (chain: A, length: 86), 100.0 (chain: B, length: 1)
1	Distance restraints	CNS/XPLOR_distance_constraints_2	OK	3527 (1)	noe	98.8 (chain: A, length: 86)
1	Dihedral angle restraints	CNS/XPLOR_dihedral_4	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%

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

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

-
X
|
X

Total number of assignments
- ¹H chemical shifts: 492
- ¹³C chemical shifts: 362
- ¹⁵N chemical shifts: 92

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	159.604
12	ARG	CZ	159.637
34	ARG	CZ	159.863
51	ARG	CZ	159.634
55	ARG	CZ	159.972
67	ARG	CZ	159.799
72	ARG	HH11	6.914
72	ARG	HH12	6.914
72	ARG	HH21	6.914
72	ARG	HH22	6.914
72	ARG	CZ	159.467
72	ARG	NH1	71.227
72	ARG	NH2	71.227

All atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	462	453	98.1
¹³C chemical shifts	364	355	97.5
¹⁵N chemical shifts	92	90	97.8

Backbone atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	177	174	98.3
¹³C chemical shifts	172	167	97.1
¹⁵N chemical shifts	84	82	97.6

Side chain atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	285	279	97.9
¹³C chemical shifts	192	188	97.9
¹⁵N chemical shifts	8	8	100.0

Methyl group atoms

Atom group	# of target shifts	# of assigned shifts	Completeness (%)
¹H chemical shifts	62	60	96.8
¹³C chemical shifts	62	60	96.8

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: 3527
- Weight of restraints: 1.0 (3527)
- Potential type of restraints: square-well-parabolic (3527)
- 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_4
- 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, Fatty Acid, Octanoyl, Polyketide