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- PDB-9q3i: Cryo-EM Structure of the Class II Cyclase Domain in the Bifunctio... -

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Basic information

Entry
Database: PDB / ID: 9q3i
TitleCryo-EM Structure of the Class II Cyclase Domain in the Bifunctional Copalyl Diphosphate Synthase from Penicillium verruculosum
ComponentsCopalyl diphosphate synthase
KeywordsLYASE / Terpene / Natural products / Cyclization
Function / homology
Function and homology information


copalyl diphosphate synthase / alcohol biosynthetic process / mycotoxin biosynthetic process / geranylgeranyl diphosphate synthase / prenyltransferase activity / isoprenoid biosynthetic process / isomerase activity / metal ion binding
Similarity search - Function
Polyprenyl synthases signature 1. / Polyprenyl synthases signature 2. / Polyprenyl synthetase, conserved site / Polyprenyl synthetase / Polyprenyl synthetase / Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid / Isoprenoid synthase domain superfamily
Similarity search - Domain/homology
Copalyl diphosphate synthase
Similarity search - Component
Biological speciesTalaromyces verruculosus (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGaynes, M.N. / Christianson, D.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM56838 United States
CitationJournal: bioRxiv / Year: 2025
Title: Cryo-EM Structure of the Cyclase Domain and Evaluation of Substrate Channeling in a Bifunctional Class II Terpene Synthase.
Authors: Matthew N Gaynes / Kollin Schultz / Eliott S Wenger / Trey A Ronnebaum / Ronen Marmorstein / David W Christianson /
Abstract: Copalyl diphosphate synthase from (PvCPS) is a bifunctional class II terpene synthase containing a prenyltransferase that produces geranylgeranyl diphosphate (GGPP) and a class II cyclase that ...Copalyl diphosphate synthase from (PvCPS) is a bifunctional class II terpene synthase containing a prenyltransferase that produces geranylgeranyl diphosphate (GGPP) and a class II cyclase that utilizes GGPP as a substrate to generate the bicyclic diterpene copalyl diphosphate. The various stereoisomers of copalyl diphosphate establish the greater family of labdane natural products, many of which have environmental and medicinal impact. Understanding structure-function relationships in class II diterpene synthases is crucial for guiding protein engineering campaigns aimed at the generation of diverse bicyclic diterpene scaffolds. However, only a limited number of structures are available for class II cyclases from bacteria, plants, and humans, and no structures are available for a class II cyclase from a fungus. Further, bifunctional class II terpene synthases have not been investigated with regard to substrate channeling between the prenyltransferase and the cyclase. Here, we report the 2.9 Å-resolution cryo-EM structure of the 63-kD class II cyclase domain from PvCPS. Comparisons with bacterial and plant copalyl diphosphate synthases reveal conserved residues that likely guide the formation of the bicyclic labdane core, but divergent catalytic dyads that mediate the final deprotonation step of catalysis. Substrate competition experiments reveal preferential GGPP transit from the PvCPS prenyltransferase to the cyclase, even when prepared as separate constructs. These results are consistent with a model in which transient prenyltransferase-cyclase association facilitates substrate channeling due to active site proximity.
History
DepositionAug 18, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 24, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Copalyl diphosphate synthase


Theoretical massNumber of molelcules
Total (without water)110,9961
Polymers110,9961
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Copalyl diphosphate synthase / CPS / Bifunctional diterpene synthase PvCPS


Mass: 110996.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Talaromyces verruculosus (fungus) / Gene: PvCPS / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A348FUE1, copalyl diphosphate synthase, geranylgeranyl diphosphate synthase
Has protein modificationN

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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: Copalyl diphosphate synthase / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.108 MDa / Experimental value: NO
Source (natural)Organism: Talaromyces verruculosus (fungus)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
31 mMTCEPC9H15O6P1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / 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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 3.29 sec. / Electron dose: 33 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4100

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
4cryoSPARC4.6.2CTF correction
7UCSF ChimeraX1.4model fitting
11RELION5classification
12RELION53D reconstruction
13PHENIX1.20.1_4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1686323
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 381049 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 2.9 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033712
ELECTRON MICROSCOPYf_angle_d0.475064
ELECTRON MICROSCOPYf_dihedral_angle_d11.9241216
ELECTRON MICROSCOPYf_chiral_restr0.037605
ELECTRON MICROSCOPYf_plane_restr0.003627

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