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- EMDB-72194: Cryo-EM Structure of the Class II Cyclase Domain in the Bifunctio... -

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Basic information

Entry
Database: EMDB / ID: EMD-72194
TitleCryo-EM Structure of the Class II Cyclase Domain in the Bifunctional Copalyl Diphosphate Synthase from Penicillium verruculosum
Map dataUnsharpened map Class II Cyclase Domain in the Bifunctional Copalyl Diphosphate Synthase from Penicillium verruculosum
Sample
  • Complex: Copalyl diphosphate synthase
    • Protein or peptide: Copalyl diphosphate synthase
KeywordsTerpene / Natural products / Cyclization / LYASE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGaynes MN / Christianson DW
Funding support United States, 1 items
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, 2025-
Header (metadata) releaseSep 24, 2025-
Map releaseSep 24, 2025-
UpdateSep 24, 2025-
Current statusSep 24, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_72194.png

Downloads & links

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Map

FileDownload / File: emd_72194.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map Class II Cyclase Domain in the Bifunctional Copalyl Diphosphate Synthase from Penicillium verruculosum
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 180 pix.
= 192.6 Å		1.07 Å/pix.
x 180 pix.
= 192.6 Å		1.07 Å/pix.
x 180 pix.
= 192.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.02532338 - 0.05223109
Average (Standard dev.)0.000071089475 (±0.0014108769)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 192.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened map

Fileemd_72194_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_72194_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_72194_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Copalyl diphosphate synthase

EntireName: Copalyl diphosphate synthase
Components
  • Complex: Copalyl diphosphate synthase
    • Protein or peptide: Copalyl diphosphate synthase

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Supramolecule #1: Copalyl diphosphate synthase

SupramoleculeName: Copalyl diphosphate synthase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Talaromyces verruculosus (fungus)
Molecular weightTheoretical: 108 KDa

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Macromolecule #1: Copalyl diphosphate synthase

MacromoleculeName: Copalyl diphosphate synthase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: copalyl diphosphate synthase
Source (natural)Organism: Talaromyces verruculosus (fungus)
Molecular weightTheoretical: 110.996031 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGENLYFQG HMASMSPMDL QESAAALVRQ LGERVEDRRG FGFMSPAIYD TAWVSMISKT IDDQKTWLFA ECFQYILSH QLEDGGWAMY ASEIDAILNT SASLLSLKRH LSNPYQITSI TQEDLSARIN RAQNALQKLL NEWNVDSTLH V GFEILVPA ...String:
MGSSHHHHHH SSGENLYFQG HMASMSPMDL QESAAALVRQ LGERVEDRRG FGFMSPAIYD TAWVSMISKT IDDQKTWLFA ECFQYILSH QLEDGGWAMY ASEIDAILNT SASLLSLKRH LSNPYQITSI TQEDLSARIN RAQNALQKLL NEWNVDSTLH V GFEILVPA LLRYLEDEGI AFAFSGRERL LEIEKQKLSK FKAQYLYLPI KVTALHSLEA FIGAIEFDKV SHHKVSGAFM AS PSSTAAY MMHATQWDDE CEDYLRHVIA HASGKGSGGV PSAFPSTIFE SVWPLSTLLK VGYDLNSAPF IEKIRSYLHD AYI AEKGIL GFTPFVGADA DDTATTILVL NLLNQPVSVD AMLKEFEEEH HFKTYSQERN PSFSANCNVL LALLYSQEPS LYSA QIEKA IRFLYKQFTD SEMDVRDKWN LSPYYSWMLM TQAITRLTTL QKTSKLSTLR DDSISKGLIS LLFRIASTVV KDQKP GGSW GTRASKEETA YAVLILTYAF YLDEVTESLR HDIKIAIENG CSFLSERTMQ SDSEWLWVEK VTYKSEVLSE AYILAA LKR AADLPDENAE AAPVINGIST NGFEHTDRIN GKLKVNGTNG TNGSHETNGI NGTHEIEQIN GVNGTNGHSD VPHDTNG WV EEPTAINETN GHYVNGTNHE TPLTNGISNG DSVSVHTDHS DSYYQRSDWT ADEEQILLGP FDYLESLPGK NMRSQLIQ S FNTWLKVPTE SLDVIIKVIS MLHTASLLID DIQDQSILRR GQPVAHSIFG TAQAMNSGNY VYFLALREVQ KLQNPKAIS IYVDSLIDLH RGQGMELFWR DSLMCPTEEQ YLDMVANKTG GLFCLAIQLM QAEATIQVDF IPLVRLLGII FQICDDYLNL KSTAYTDNK GLCEDLTEGK FSFPIIHSIR SNPGNRQLIN ILKQKPREDD IKRYALSYME STNSFEYTRG VVRKLKTEAI D TIQGLEKH GLEENIGIRK ILARMSLEL

UniProtKB: Copalyl diphosphate synthase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
1.0 mMC9H15O6PTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 4100 / Average exposure time: 3.29 sec. / Average electron dose: 33.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1686323
CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: AlphaFold3 model of the PvCPS class II cyclsae domain
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5.0) / Number images used: 381049
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-9q3i:
Cryo-EM Structure of the Class II Cyclase Domain in the Bifunctional Copalyl Diphosphate Synthase from Penicillium verruculosum

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