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- PDB-6fij: Structure of the loading/condensing region (SAT-KS-MAT) of the ce... -

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Entry
Database: PDB / ID: 6fij
TitleStructure of the loading/condensing region (SAT-KS-MAT) of the cercosporin fungal non-reducing polyketide synthase (NR-PKS) CTB1
ComponentsPolyketide synthase
KeywordsBIOSYNTHETIC PROTEIN / NR-PKS / PKS / iPKS / iterative PKS / non-reducing / SAT / starter acyl / condensing / loading / polyketide / fungal
Function / homology
Function and homology information


secondary metabolite biosynthetic process / fatty acid synthase activity / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / fatty acid biosynthetic process
Similarity search - Function
Polyketide product template domain / Malonyl-Coenzyme A Acyl Carrier Protein, domain 2 / Starter unit:ACP transacylase / Starter unit:ACP transacylase in aflatoxin biosynthesis / Malonyl-Coenzyme A Acyl Carrier Protein; domain 2 / Polyketide synthase, thioesterase domain / Thioesterase / CurL-like, PKS C-terminal / Thioesterase / Thioesterase domain ...Polyketide product template domain / Malonyl-Coenzyme A Acyl Carrier Protein, domain 2 / Starter unit:ACP transacylase / Starter unit:ACP transacylase in aflatoxin biosynthesis / Malonyl-Coenzyme A Acyl Carrier Protein; domain 2 / Polyketide synthase, thioesterase domain / Thioesterase / CurL-like, PKS C-terminal / Thioesterase / Thioesterase domain / Polyketide synthase, dehydratase domain superfamily / : / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / Malonyl-CoA ACP transacylase, ACP-binding / : / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase domain superfamily / Acyl transferase/acyl hydrolase/lysophospholipase / Thiolase/Chalcone synthase / Peroxisomal Thiolase; Chain A, domain 1 / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / Ketosynthase family 3 (KS3) domain profile. / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / Polyketide synthase, beta-ketoacyl synthase domain / Beta-ketoacyl synthase, N-terminal domain / Beta-ketoacyl synthase, C-terminal domain / Thiolase-like / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Alpha/Beta hydrolase fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
2,3-DIHYDROXY-1,4-DITHIOBUTANE / Non-reducing polyketide synthase CTB1
Similarity search - Component
Biological speciesCercospora nicotianae (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.77 Å
AuthorsHerbst, D.A. / Jakob, R.P. / Townsend, C.A. / Maier, T.
Funding support Switzerland, United States, 5items
OrganizationGrant numberCountry
Swiss National Science Foundation159696 Switzerland
Swiss National Science Foundation145023 Switzerland
Swiss National Science Foundation164074 Switzerland
Swiss National Science Foundation138262 Switzerland
National Institutes of HealthES001670 United States
CitationJournal: Nat Chem Biol / Year: 2018
Title: The structural organization of substrate loading in iterative polyketide synthases.
Authors: Dominik A Herbst / Callie R Huitt-Roehl / Roman P Jakob / Jacob M Kravetz / Philip A Storm / Jamie R Alley / Craig A Townsend / Timm Maier /
Abstract: Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an ...Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/-condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.
History
DepositionJan 18, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 21, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 4, 2018Group: Data collection / Database references / Refinement description
Category: citation / refine / Item: _citation.year / _refine.details
Revision 1.2Apr 11, 2018Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / citation ...chem_comp / citation / entity / pdbx_entity_nonpoly
Item: _chem_comp.name / _citation.journal_abbrev ..._chem_comp.name / _citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _entity.pdbx_description / _pdbx_entity_nonpoly.name
Revision 1.3Apr 25, 2018Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / citation ...chem_comp / citation / entity / pdbx_entity_nonpoly
Item: _chem_comp.name / _citation.journal_volume ..._chem_comp.name / _citation.journal_volume / _citation.page_first / _citation.page_last / _entity.pdbx_description / _pdbx_entity_nonpoly.name
Revision 1.4Jan 17, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Polyketide synthase
B: Polyketide synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)281,71418
Polymers280,6062
Non-polymers1,10816
Water6,341352
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Gel filtration, cryoEM, structure homology (all PKS are dimers)
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17530 Å2
ΔGint-28 kcal/mol
Surface area89630 Å2
MethodPISA
Unit cell
Length a, b, c (Å)108.050, 230.200, 253.800
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11B-1407-

MG

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Polyketide synthase


Mass: 140303.062 Da / Num. of mol.: 2 / Mutation: T321A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cercospora nicotianae (fungus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q6DQW3

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Non-polymers , 5 types, 368 molecules

#2: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C2H6O2
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-DTT / 2,3-DIHYDROXY-1,4-DITHIOBUTANE / 1,4-DITHIOTHREITOL


Mass: 154.251 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C4H10O2S2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 352 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.81 Å3/Da / Density % sol: 56.32 % / Description: plate-like
Crystal growTemperature: 277.15 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 0.2 M MgCl2 0.1 M Bis-Tris Propane pH 6.5 18% (v/v) PEG3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Sep 16, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.77→126.915 Å / Num. obs: 79860 / % possible obs: 99.2 % / Redundancy: 8.5 % / Biso Wilson estimate: 60.9 Å2 / CC1/2: 0.995 / Rmerge(I) obs: 0.212 / Net I/σ(I): 10.13
Reflection shellResolution: 2.77→2.84 Å / Redundancy: 5.6 % / Rmerge(I) obs: 1.938 / Mean I/σ(I) obs: 1.03 / Num. unique obs: 5448 / CC1/2: 0.433 / % possible all: 91.9

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Processing

Software
NameVersionClassification
PHENIX(1.10_2155: ???)refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2HG4, 4RO5

2hg4

4ro5


Resolution: 2.77→63.45 Å / SU ML: 0.46 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.1 / Stereochemistry target values: ML
Details: UNIDENTIFIED DIFFERENCE DENSITY AROUND THE ACTIVE SITE RESIDUES C553 AND C119, POSSIBLY DUE TO PARTIAL ACYLATION OR OXIDATION. THE DIFFERENCE DENSITY IN THE REGION AROUND RESIDUES 651 TO 661 ...Details: UNIDENTIFIED DIFFERENCE DENSITY AROUND THE ACTIVE SITE RESIDUES C553 AND C119, POSSIBLY DUE TO PARTIAL ACYLATION OR OXIDATION. THE DIFFERENCE DENSITY IN THE REGION AROUND RESIDUES 651 TO 661 INDICATES MULTIPLE DIFFERENT MAIN CHAIN CONFORMATION, WHEREAS ONLY ONE COULD ME MODELLED. BASED ON OBSERVED B-FACTORS AND THE FACT THAT CRYSTALLIZATION WAS CARRIED OUT IN THE PRESENCE OF 0.3M MG(2+) SOME SURFACE WATERS MIGHT ALSO REPRESENT PARTIALLY OCCUPIED HYDRATED MAGNESIUM IONS.
RfactorNum. reflection% reflection
Rfree0.2405 3916 4.91 %
Rwork0.2052 --
obs0.2069 79812 99.04 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.77→63.45 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms19291 0 69 352 19712
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00219940
X-RAY DIFFRACTIONf_angle_d0.49627153
X-RAY DIFFRACTIONf_dihedral_angle_d11.92512022
X-RAY DIFFRACTIONf_chiral_restr0.043085
X-RAY DIFFRACTIONf_plane_restr0.0033570
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7698-2.80360.45421320.46262366X-RAY DIFFRACTION89
2.8036-2.83910.43971270.40512551X-RAY DIFFRACTION93
2.8391-2.87640.37671250.36732608X-RAY DIFFRACTION96
2.8764-2.91580.3661290.35362699X-RAY DIFFRACTION99
2.9158-2.95750.3861250.3222707X-RAY DIFFRACTION99
2.9575-3.00160.30451430.30392673X-RAY DIFFRACTION100
3.0016-3.04850.32491400.28642698X-RAY DIFFRACTION100
3.0485-3.09850.33331410.28472742X-RAY DIFFRACTION100
3.0985-3.15190.32091420.27572694X-RAY DIFFRACTION100
3.1519-3.20920.35121440.2692705X-RAY DIFFRACTION100
3.2092-3.2710.30811420.2582721X-RAY DIFFRACTION100
3.271-3.33770.30091400.25382688X-RAY DIFFRACTION100
3.3377-3.41030.28561220.24452721X-RAY DIFFRACTION100
3.4103-3.48960.25521450.22822754X-RAY DIFFRACTION100
3.4896-3.57690.2351450.21492686X-RAY DIFFRACTION100
3.5769-3.67360.23821530.21192714X-RAY DIFFRACTION100
3.6736-3.78170.23971540.19032719X-RAY DIFFRACTION100
3.7817-3.90370.19931310.19352732X-RAY DIFFRACTION100
3.9037-4.04320.22721370.17792732X-RAY DIFFRACTION100
4.0432-4.20510.20671450.17492731X-RAY DIFFRACTION100
4.2051-4.39640.23641540.15492730X-RAY DIFFRACTION100
4.3964-4.62810.18141380.15292746X-RAY DIFFRACTION100
4.6281-4.9180.17181490.1492756X-RAY DIFFRACTION100
4.918-5.29750.17761350.162761X-RAY DIFFRACTION100
5.2975-5.83030.21561500.17562749X-RAY DIFFRACTION100
5.8303-6.67320.21071440.18422796X-RAY DIFFRACTION100
6.6732-8.40440.20031360.16752814X-RAY DIFFRACTION100
8.4044-63.46660.23091480.192903X-RAY DIFFRACTION99
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.23050.05160.0682.5314-0.80640.57560.0381-0.05390.13420.0927-0.01260.3934-0.0409-0.027-0.04280.5078-0.11810.00210.5741-0.08190.5185-6.268185.6464-22.2192
21.76550.82840.22152.66640.35661.35160.2133-0.27880.37460.4609-0.16840.1906-0.11410.1333-0.04770.4688-0.10590.02190.4324-0.00980.3535-2.719548.3417-7.6392
32.38190.61641.20240.54220.19841.14190.1742-0.28660.05550.0945-0.12310.24640.1814-0.2935-0.04940.4609-0.0506-0.01340.43890.01290.5061-43.376426.2176-29.1474
42.05591.01960.6411.98460.93592.2877-0.07810.08110.2811-0.00720.0838-0.0026-0.29880.2446-0.01790.4555-0.0457-0.16650.44180.03830.6883-33.083158.356-50.3213
51.25510.34040.34191.09770.17740.90350.02470.1637-0.0968-0.1140.0233-0.05450.08470.0276-0.05890.3357-0.0063-0.02950.4102-0.00720.268710.873646.3087-35.6
60.48190.25440.13243.00080.25710.1008-0.04420.09520.10290.20350.0265-0.6904-0.1190.20670.01270.5035-0.0987-0.09890.59180.06820.575734.380991.4807-25.4051
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 5 through 441 )
2X-RAY DIFFRACTION2chain 'A' and (resid 442 through 805 )
3X-RAY DIFFRACTION3chain 'A' and (resid 806 through 1288 )
4X-RAY DIFFRACTION4chain 'B' and (resid 5 through 337 )
5X-RAY DIFFRACTION5chain 'B' and (resid 338 through 823 )
6X-RAY DIFFRACTION6chain 'B' and (resid 824 through 1288 )

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