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- EMDB-42855: Cryo-EM structure of the unliganded hexameric prenyltransferase i... -

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Basic information

Entry
Database: EMDB / ID: EMD-42855
TitleCryo-EM structure of the unliganded hexameric prenyltransferase in bifunctional copalyl diphosphate synthase from Penicillium fellutanum with an open conformation, reconstruction in C1
Map data
Sample
  • Complex: Hexameric prenyltransferase from the bifunctional copalyl diphosphate synthase of Penicillium fellutanum
    • Protein or peptide: Copalyl diphosphate synthase
Keywordsterpene / biosynthesis / enzyme / TRANSFERASE
Biological speciesPenicillium fellutanum ATCC 48694 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.94 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: 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, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateJan 24, 2024-
Current statusJan 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42855.png

Downloads & links

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Map

FileDownload / File: emd_42855.map.gz / Format: CCP4 / Size: 729 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.55 Å/pix.
x 576 pix.
= 316.8 Å		0.55 Å/pix.
x 576 pix.
= 316.8 Å		0.55 Å/pix.
x 576 pix.
= 316.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.55 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.066
Minimum - Maximum-0.001773588 - 1.6670055
Average (Standard dev.)0.0010407213 (±0.023811849)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions576576576
Spacing576576576
CellA=B=C: 316.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened C1 map of the hexameric PfCPS prenyltransferase...

Fileemd_42855_additional_1.map
AnnotationUnsharpened C1 map of the hexameric PfCPS prenyltransferase in non-uniform refinement
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Half map: #1

Fileemd_42855_half_map_1.map
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Half map: #2

Fileemd_42855_half_map_2.map
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Sample components

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Entire : Hexameric prenyltransferase from the bifunctional copalyl diphosp...

EntireName: Hexameric prenyltransferase from the bifunctional copalyl diphosphate synthase of Penicillium fellutanum
Components
  • Complex: Hexameric prenyltransferase from the bifunctional copalyl diphosphate synthase of Penicillium fellutanum
    • Protein or peptide: Copalyl diphosphate synthase

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Supramolecule #1: Hexameric prenyltransferase from the bifunctional copalyl diphosp...

SupramoleculeName: Hexameric prenyltransferase from the bifunctional copalyl diphosphate synthase of Penicillium fellutanum
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Penicillium fellutanum ATCC 48694 (fungus)
Molecular weightTheoretical: 634.002 KDa

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Macromolecule #1: Copalyl diphosphate synthase

MacromoleculeName: Copalyl diphosphate synthase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Penicillium fellutanum ATCC 48694 (fungus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGENLYFQG HMASMESASL DQSAALLVKE LTEHIDDSNG LGFMSPAIYD TAWVSMIKKT DNDQTFWLFP KSFHYILENQ LENGGWVTYA SEIDGILNTS ASLLSLKRHF DLPLQISTES QTSMENRIRK ATDALRVLLR TWDVDATLHV GFEILVPALL ...String:
MGSSHHHHHH SSGENLYFQG HMASMESASL DQSAALLVKE LTEHIDDSNG LGFMSPAIYD TAWVSMIKKT DNDQTFWLFP KSFHYILENQ LENGGWVTYA SEIDGILNTS ASLLSLKRHF DLPLQISTES QTSMENRIRK ATDALRVLLR TWDVDATLHV GFEILVPALL DYLQVEGLTF DFPGRDKLFQ IRDQKLSRFK PEFIYAPFQT TALHSLEAFI GLIDFDRVQH HKVRGSFMAS PSSTAAVLMN ATEWDIDCEE YIRHVIEHAS GKASGGVPSA FPSTIFEITW TLSTLLKAGF NLSSNDSSNV QKACSYLLGV LTAEKGAIGF VPSVCADADD TAKTILVLSL LRENVLPDGM LKAFEVENHF KTYPLERDPS FSANCNVLLA LLHLENPSQY TLQIEKATRF LYTHFRESNL NVRDKWNLSP FYSWMLMAQA IARLDELCKG SQLKNLHDHV ESDLIPLLQE MTVSVMHQQN KDGSWGTKLS KEETAYAVLM LTYAVSFEAL VGPRRQIRNA IEEGCLFLRS GKDATSERLW VEKVTYESQM LSRAYTLAAL KNALDVLKKD DMKIAFIGNS VNESFTEIEV VNGKNVTEPS SNTYDLKEVK MDKAEFTPPD TPPRSKSTSV DPIEEAICAH ENRGAISDHT SRAIDLCRNP PLWGTDQEQT LLGPFEYLES IPGKNIRSQF IEAFNTWLQI PQDHLQIVGK VISMLHTASL LVDDIEDNSL LRRGQPVAHS IFGTAQTFNS GNYVYFLALQ EVQKLNSPRA ISIFVDALTQ LHRGQGMDVF WRDSLICPTE EEYLDMVANK TGALFCLAIE LLQIKSTVQL DFLPLVRLLG IIFQICDDYL NLKSTNYTQK KGLCEDITEG KFSFPIIHSI RTKPGNRQLI NVLRQKSKED DVKRFALAYM ESTQSFDYTR DFVKILNGEA LRMIEDLEQQ GLHRNIEIRN ILARMSLEQ

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
50.0 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 I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 12467 / Average exposure time: 2.15 sec. / Average electron dose: 43.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination 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: 3639075
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 267283
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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