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- PDB-7m7f: 6-Deoxyerythronolide B synthase (DEBS) module 1 in complex with a... -

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Basic information

Entry
Database: PDB / ID: 7m7f
Title6-Deoxyerythronolide B synthase (DEBS) module 1 in complex with antibody fragment 1B2: State 1
Components
  • 1B2 (heavy chain)
  • 1B2 (light chain)
  • EryAI,6-deoxyerythronolide-B synthase EryA3, modules 5 and 6 chimera
KeywordsBIOSYNTHETIC PROTEIN/IMMUNE SYSTEM / polyketide synthase / antibody fragment / BIOSYNTHETIC PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


6-deoxyerythronolide-B synthase / erythronolide synthase activity / macrolide biosynthetic process / phosphopantetheine binding / 3-oxoacyl-[acyl-carrier-protein] synthase activity / antibiotic biosynthetic process / fatty acid biosynthetic process
Similarity search - Function
Polyketide synthase, docking domain superfmaily / Polyketide synthase, thioesterase domain / Thioesterase / Polyketide synthase, docking domain / Erythronolide synthase docking domain / PKS_PP_betabranch / Thioesterase / Thioesterase domain / Polyketide synthase, ketoreductase domain / KR domain ...Polyketide synthase, docking domain superfmaily / Polyketide synthase, thioesterase domain / Thioesterase / Polyketide synthase, docking domain / Erythronolide synthase docking domain / PKS_PP_betabranch / Thioesterase / Thioesterase domain / Polyketide synthase, ketoreductase domain / KR domain / Polyketide synthase, C-terminal extension / Ketoacyl-synthetase C-terminal extension / Malonyl-CoA ACP transacylase, ACP-binding / PKS_KR / Acyl transferase domain superfamily / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase/acyl hydrolase/lysophospholipase / Ketosynthase family 3 (KS3) domain profile. / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / Beta-ketoacyl synthase / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / Polyketide synthase, beta-ketoacyl synthase domain / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / Beta-ketoacyl synthase, N-terminal domain / Beta-ketoacyl synthase, C-terminal domain / Phosphopantetheine attachment site / Thiolase-like / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Alpha/Beta hydrolase fold / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Chem-PN7 / 6-deoxyerythronolide-B synthase EryA3, modules 5 and 6 / EryAI
Similarity search - Component
Biological speciesSaccharopolyspora erythraea (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsCogan, D.P. / Zhang, K. / Chiu, W. / Khosla, C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
CitationJournal: Science / Year: 2021
Title: Mapping the catalytic conformations of an assembly-line polyketide synthase module.
Authors: Dillon P Cogan / Kaiming Zhang / Xiuyuan Li / Shanshan Li / Grigore D Pintilie / Soung-Hun Roh / Charles S Craik / Wah Chiu / Chaitan Khosla /
Abstract: Assembly-line polyketide synthases, such as the 6-deoxyerythronolide B synthase (DEBS), are large enzyme factories prized for their ability to produce specific and complex polyketide products. By ...Assembly-line polyketide synthases, such as the 6-deoxyerythronolide B synthase (DEBS), are large enzyme factories prized for their ability to produce specific and complex polyketide products. By channeling protein-tethered substrates across multiple active sites in a defined linear sequence, these enzymes facilitate programmed small-molecule syntheses that could theoretically be harnessed to access countless polyketide product structures. Using cryogenic electron microscopy to study DEBS module 1, we present a structural model describing this substrate-channeling phenomenon. Our 3.2- to 4.3-angstrom-resolution structures of the intact module reveal key domain-domain interfaces and highlight an unexpected module asymmetry. We also present the structure of a product-bound module that shines light on a recently described “turnstile” mechanism for transient gating of active sites along the assembly line.
History
DepositionMar 28, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
B: EryAI,6-deoxyerythronolide-B synthase EryA3, modules 5 and 6 chimera
A: EryAI,6-deoxyerythronolide-B synthase EryA3, modules 5 and 6 chimera
C: 1B2 (heavy chain)
D: 1B2 (light chain)
E: 1B2 (heavy chain)
F: 1B2 (light chain)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)481,7877
Polymers481,4296
Non-polymers3581
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein EryAI,6-deoxyerythronolide-B synthase EryA3, modules 5 and 6 chimera / DEBS (3)M1TE


Mass: 188550.891 Da / Num. of mol.: 2
Fragment: EryA1 (UNP residues 225-2010) + EryA3 (UNP residues 2896-3172)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharopolyspora erythraea (bacteria) / Gene: eryAI, eryA / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q5UNP6, UniProt: Q03133, 6-deoxyerythronolide-B synthase
#2: Antibody 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)
#3: Antibody 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)
#4: Chemical ChemComp-PN7 / N~3~-[(2S)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-N-(2-sulfanylethyl)-beta-alaninamide


Mass: 358.348 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H23N2O7PS
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

ComponentName: Complex between DEBS (3)M1TE and antibody fragment 1B2
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.48 MDa / Experimental value: NO
Source (natural)Organism: Saccharopolyspora erythraea (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMcitric acidC6H8O71
2100 mMsodium chlorideNaClSodium chloride1
310 mMHEPESC8H18N2O4S1
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8.5 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 10234

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategory
1EMAN22.3particle selection
2EPUimage acquisition
4CTFFINDCTF correction
11cryoSPARCV2final Euler assignment
12cryoSPARCv2classification
13cryoSPARCv23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1406538
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 172352 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01224473
ELECTRON MICROSCOPYf_angle_d1.69433299
ELECTRON MICROSCOPYf_dihedral_angle_d18.7148817
ELECTRON MICROSCOPYf_chiral_restr0.0933754
ELECTRON MICROSCOPYf_plane_restr0.0094415

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