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- PDB-7s6c: CryoEM structure of modular PKS holo-Lsd14 stalled at the condens... -

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Basic information

Entry
Database: PDB / ID: 7s6c
TitleCryoEM structure of modular PKS holo-Lsd14 stalled at the condensation step and bound to antibody fragment 1B2, composite structure
Components
  • 6-deoxyerythronolide-B synthase EryA2, modules 3 and 4, Lsd14 Polyketide synthase fusion
  • Fab 1B2 heavy chain
  • Fab 1B2 light chain
KeywordsBIOSYNTHETIC PROTEIN / Modular polyketide synthase / ketosynthase / ketoreductase / acyl carrier protein
Function / homology
Function and homology information


6-deoxyerythronolide-B synthase / erythronolide synthase activity / macrolide biosynthetic process / DIM/DIP cell wall layer assembly / fatty acid synthase activity / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process / oxidoreductase activity / plasma membrane / cytoplasm
Similarity search - Function
Erythronolide synthase, docking / Erythronolide synthase, docking domain superfamily / Erythronolide synthase, docking / Polyketide synthase dimerisation element domain / Polyketide synthase dimerisation element domain / Polyketide synthase, docking domain superfmaily / Polyketide synthase extender module SpnB, Rossmann fold domain / Polyketide synthase, docking domain / Erythronolide synthase docking domain / Zinc-binding dehydrogenase ...Erythronolide synthase, docking / Erythronolide synthase, docking domain superfamily / Erythronolide synthase, docking / Polyketide synthase dimerisation element domain / Polyketide synthase dimerisation element domain / Polyketide synthase, docking domain superfmaily / Polyketide synthase extender module SpnB, Rossmann fold domain / Polyketide synthase, docking domain / Erythronolide synthase docking domain / Zinc-binding dehydrogenase / : / Polyketide synthase dehydratase N-terminal domain / Polyketide synthase, dehydratase domain / PKS_DH / : / Polyketide synthase dehydratase domain / Polyketide synthase, dehydratase domain superfamily / : / Polyketide and metazoan fatty acid synthase dehydratase (PKS/mFAS DH) domain profile. / PKS_PP_betabranch / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Polyketide synthase, ketoreductase domain / KR domain / 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 / Polyketide synthase, enoylreductase domain / Enoylreductase / Alcohol dehydrogenase, N-terminal / Alcohol dehydrogenase GroES-like domain / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / PKS_KR / 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 / GroES-like superfamily / Thiolase-like / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
2'-MONOPHOSPHOADENOSINE-5'-DIPHOSPHATE / 4'-PHOSPHOPANTETHEINE / Putative polyketide synthase / 6-deoxyerythronolide-B synthase EryA2, modules 3 and 4
Similarity search - Component
Biological speciesSaccharopolyspora erythraea (bacteria)
Streptomyces lasalocidi (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsBagde, S.R. / Kim, C.-Y. / Fromme, J.C.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM136258 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM138990 United States
National Science Foundation (NSF, United States)DMR-1719875 United States
CitationJournal: Science / Year: 2021
Title: Modular polyketide synthase contains two reaction chambers that operate asynchronously.
Authors: Saket R Bagde / Irimpan I Mathews / J Christopher Fromme / Chu-Young Kim /
Abstract: Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and β-keto ...Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and β-keto group modification reactions. We determined the 2.4-angstrom-resolution x-ray crystal structure and the 3.1-angstrom-resolution cryo–electron microscopy structure of the Lsd14 polyketide synthase, stalled at the transacylation and condensation steps, respectively. These structures revealed how the constituent domains are positioned relative to each other, how they rearrange depending on the step in the reaction cycle, and the specific interactions formed between the domains. Like the evolutionarily related mammalian fatty acid synthase, Lsd14 contains two reaction chambers, but only one chamber in Lsd14 has the full complement of catalytic domains, indicating that only one chamber produces the polyketide product at any given time.
History
DepositionSep 13, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 3, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 17, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-24868
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: 6-deoxyerythronolide-B synthase EryA2, modules 3 and 4, Lsd14 Polyketide synthase fusion
B: 6-deoxyerythronolide-B synthase EryA2, modules 3 and 4, Lsd14 Polyketide synthase fusion
C: 6-deoxyerythronolide-B synthase EryA2, modules 3 and 4, Lsd14 Polyketide synthase fusion
D: 6-deoxyerythronolide-B synthase EryA2, modules 3 and 4, Lsd14 Polyketide synthase fusion
E: Fab 1B2 heavy chain
F: Fab 1B2 light chain
G: Fab 1B2 light chain
H: Fab 1B2 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)801,53510
Polymers800,6708
Non-polymers8662
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21970 Å2
ΔGint-122 kcal/mol
Surface area118300 Å2

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Components

#1: Protein
6-deoxyerythronolide-B synthase EryA2, modules 3 and 4, Lsd14 Polyketide synthase fusion / DEBS 2 / 6-deoxyerythronolide B synthase II / Erythronolide synthase / ORF B


Mass: 174085.734 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharopolyspora erythraea (bacteria), (gene. exp.) Streptomyces lasalocidi (bacteria)
Gene: eryA, lsd14 / Production host: Escherichia coli (E. coli) / Strain (production host): BAP1
References: UniProt: Q03132, UniProt: B6ZK67, 6-deoxyerythronolide-B synthase
#2: Antibody Fab 1B2 heavy chain


Mass: 26447.611 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
#3: Antibody Fab 1B2 light chain


Mass: 25715.832 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
#4: Chemical ChemComp-ATR / 2'-MONOPHOSPHOADENOSINE-5'-DIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3
#5: Chemical ChemComp-PNS / 4'-PHOSPHOPANTETHEINE


Mass: 358.348 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H23N2O7PS / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Dimeric holo-Lsd14 containing docking domains from DEBS module 3 (holo-Lsd14-DD*) bound to two copies each of Fab 1B2 light and heavy chains and NADP, treated with 2-Acetaminoethyl-thio-3-oxobutanoateCOMPLEX#1-#30RECOMBINANT
2Polyketide synthaseCOMPLEX1RECOMBINANT
3Fab 1B2 heavy chain, Fab 1B2 light chainCOMPLEX1RECOMBINANT
Molecular weightValue: 0.45 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Streptomyces lasalocidi (bacteria)324833
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Escherichia coli (E. coli)562
23Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameBuffer-ID
1100 mMsodium chloride1
2100 mMcitrate1
310 mMHEPES1
41 mM2-Acetaminoethyl-thio-3-oxobutanoate1
50.4 mMmethylmalonyl-CoA1
60.8 mMNADP1
70.8 mMTCEP1
SpecimenConc.: 4.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Size exclusion chromatography purified monodisperse holo-Lsd14-DD* + 1B2 complex was first incubated with KS substrate analog (2-Acetaminoethyl-thio-3-oxobutanoate) and TCEP at 30C for 20 ...Details: Size exclusion chromatography purified monodisperse holo-Lsd14-DD* + 1B2 complex was first incubated with KS substrate analog (2-Acetaminoethyl-thio-3-oxobutanoate) and TCEP at 30C for 20 minutes, followed by addition of NADP and methylmalonyl-CoA. The sample was further incubated at 30C for 15 minutes before applying to cryoEM grids.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Blot for 5 seconds before plunging in liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 79000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.2 sec. / Electron dose: 53 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1278 / Details: Images were collected as 50 frame movies.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2.0particle selectionBlob picker and Template Picker
2EPUimage acquisition
4cryoSPARC3.2.0CTF correctionPatch CTF estimation (multi)
5RELION3.1CTF correction
11cryoSPARC3.2.0initial Euler assignment
12RELION3.1final Euler assignment
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 397539
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 397539
Details: Composite map was generated by combining the unsharpened focused maps for KS-KS'-ACP domains (EMD-24863), LDAT domains (EMD-24864), LD'AT' domains (EMD-24865), KR domain (EMD-24866), Fab + ...Details: Composite map was generated by combining the unsharpened focused maps for KS-KS'-ACP domains (EMD-24863), LDAT domains (EMD-24864), LD'AT' domains (EMD-24865), KR domain (EMD-24866), Fab + DD* (EMD-24867), and the unsharpened consensus map (EMD-24862) using Combine Focused Maps in Phenix.
Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Model was manually rebuilt in Coot and phenix.real_space_refine was used for refinement.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
17S6B

7s6b

1
26C9U

6c9u

H1
36C9U

6c9u

L1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 136.06 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003324478
ELECTRON MICROSCOPYf_angle_d0.66633398
ELECTRON MICROSCOPYf_chiral_restr0.04293818
ELECTRON MICROSCOPYf_plane_restr0.00474374
ELECTRON MICROSCOPYf_dihedral_angle_d4.86193456

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