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- PDB-8v0f: Cryo-EM structure of the unliganded hexameric prenyltransferase i... -

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Basic information

Entry
Database: PDB / ID: 8v0f
TitleCryo-EM structure of the unliganded hexameric prenyltransferase in bifunctional copalyl diphosphate synthase from Penicillium fellutanum with an open conformation
ComponentsCopalyl diphosphate synthase
KeywordsTRANSFERASE / terpene / biosynthesis / enzyme
Biological speciesPenicillium fellutanum ATCC 48694 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.81 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: J Struct Biol / Year: 2024
Title: Structure of the prenyltransferase in bifunctional copalyl diphosphate synthase from Penicillium fellutanum reveals an open hexamer conformation.
Authors: Matthew N Gaynes / Trey A Ronnebaum / Kollin Schultz / Jacque L Faylo / Ronen Marmorstein / David W Christianson /
Abstract: Copalyl diphosphate synthase from Penicillium fellutanum (PfCPS) is an assembly-line terpene synthase that contains both prenyltransferase and class II cyclase activities. The prenyltransferase ...Copalyl diphosphate synthase from Penicillium fellutanum (PfCPS) is an assembly-line terpene synthase that contains both prenyltransferase and class II cyclase activities. The prenyltransferase catalyzes processive chain elongation reactions using dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate to yield geranylgeranyl diphosphate, which is then utilized as a substrate by the class II cyclase domain to generate copalyl diphosphate. Here, we report the 2.81 Å-resolution cryo-EM structure of the hexameric prenyltransferase of full-length PfCPS, which is surrounded by randomly splayed-out class II cyclase domains connected by disordered polypeptide linkers. The hexamer can be described as a trimer of dimers; surprisingly, one of the three dimer-dimer interfaces is separated to yield an open hexamer conformation, thus breaking the D3 symmetry typically observed in crystal structures of other prenyltransferase hexamers such as wild-type human GGPP synthase (hGGPPS). Interestingly, however, an open hexamer conformation was previously observed in the crystal structure of D188Y hGGPPS, apparently facilitated by hexamer-hexamer packing in the crystal lattice. The cryo-EM structure of the PfCPS prenyltransferase hexamer is the first to reveal that an open conformation can be achieved even in the absence of a point mutation or interaction with another hexamer. Even though PfCPS octamers are not detected, we suggest that the open hexamer conformation represents an intermediate in the hexamer-octamer equilibrium for those prenyltransferases that do exhibit oligomeric heterogeneity.
History
DepositionNov 17, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Copalyl diphosphate synthase
B: Copalyl diphosphate synthase
C: Copalyl diphosphate synthase
D: Copalyl diphosphate synthase
E: Copalyl diphosphate synthase
F: Copalyl diphosphate synthase


Theoretical massNumber of molelcules
Total (without water)650,9046
Polymers650,9046
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Copalyl diphosphate synthase


Mass: 108484.070 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Penicillium fellutanum ATCC 48694 (fungus)
Production host: Escherichia coli BL21(DE3) (bacteria)

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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: Hexameric prenyltransferase from the bifunctional copalyl diphosphate synthase of Penicillium fellutanum
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.634002 MDa / Experimental value: NO
Source (natural)Organism: Penicillium fellutanum ATCC 48694 (fungus)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
250 mMsodium chlorideNaCl1
31 mMTCEPC9H15O6P1
SpecimenConc.: 0.5 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 I / 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: FEI TITAN 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 / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 2.15 sec. / Electron dose: 43 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 12467

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
4cryoSPARC4.2.1CTF correction
7UCSF ChimeraX1.4model fitting
11cryoSPARC4.2.1classification
12cryoSPARC4.2.13D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3639075
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 267283 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingSource name: AlphaFold / Type: in silico model

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