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- EMDB-21100: CYP102A1-A82F-D12-closed -

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Basic information

Entry
Database: EMDB / ID: EMD-21100
TitleCYP102A1-A82F-D12-closed
Map dataDeletion variant of CYP102A1 in closed state
Sample
  • Complex: Deletion variant of cytochrome P450 CYP102A1 enzyme
    • Protein or peptide: Cytochrome P450 102A1 (BM3)
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsSu M / Chakraborty S / Osawa Y / Zhang H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM077430 United States
CitationJournal: J Biol Chem / Year: 2020
Title: Cryo-EM reveals the architecture of the dimeric cytochrome P450 CYP102A1 enzyme and conformational changes required for redox partner recognition.
Authors: Min Su / Sumita Chakraborty / Yoichi Osawa / Haoming Zhang /
Abstract: Cytochrome P450 family 102 subfamily A member 1 (CYP102A1) is a self-sufficient flavohemeprotein and a highly active bacterial enzyme capable of fatty acid hydroxylation at a >3,000 min turnover rate. ...Cytochrome P450 family 102 subfamily A member 1 (CYP102A1) is a self-sufficient flavohemeprotein and a highly active bacterial enzyme capable of fatty acid hydroxylation at a >3,000 min turnover rate. The CYP102A1 architecture has been postulated to be responsible for its extraordinary catalytic prowess. However, the structure of a functional full-length CYP102A1 enzyme remains to be determined. Herein, we used a cryo-EM single-particle approach, revealing that full-length CYP102A1 forms a homodimer in which both the heme and FAD domains contact each other. The FMN domain of one monomer was located close to the heme domain of the other monomer, exhibiting a configuration. Moreover, full-length CYP102A1 is highly dynamic, existing in multiple conformational states, including open and closed states. In the closed state, the FMN domain closely contacts the FAD domain, whereas in the open state, one of the FMN domains rotates away from its FAD domain and traverses to the heme domain of the other monomer. This structural arrangement and conformational dynamics may facilitate rapid intraflavin and FMN-to-heme electron transfers (ETs). Results with a variant having a 12-amino-acid deletion in the CYP102A1 linker region, connecting the catalytic heme and the diflavin reductase domains, further highlighted the importance of conformational dynamics in the ET process. Cryo-EM revealed that the Δ12 variant homodimer is conformationally more stable and incapable of FMN-to-heme ET. We conclude that closed-to-open alternation is crucial for redox partner recognition and formation of an active ET complex for CYP102A1 catalysis.
History
DepositionDec 11, 2019-
Header (metadata) releaseJan 15, 2020-
Map releaseJan 15, 2020-
UpdateFeb 19, 2020-
Current statusFeb 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21100.png

Downloads & links

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Map

FileDownload / File: emd_21100.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeletion variant of CYP102A1 in closed state
Voxel sizeX=Y=Z: 1.01 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-1.3195015 - 3.1871064
Average (Standard dev.)0.00087288447 (±0.08016988)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 363.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.011.011.01
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z363.600363.600363.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-1.3203.1870.001

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Supplemental data

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Sample components

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Entire : Deletion variant of cytochrome P450 CYP102A1 enzyme

EntireName: Deletion variant of cytochrome P450 CYP102A1 enzyme
Components
  • Complex: Deletion variant of cytochrome P450 CYP102A1 enzyme
    • Protein or peptide: Cytochrome P450 102A1 (BM3)

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Supramolecule #1: Deletion variant of cytochrome P450 CYP102A1 enzyme

SupramoleculeName: Deletion variant of cytochrome P450 CYP102A1 enzyme / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: recombinant deletion variant
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: C41(DE3) / Recombinant plasmid: pCWori
Molecular weightExperimental: 238.8 KDa

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Macromolecule #1: Cytochrome P450 102A1 (BM3)

MacromoleculeName: Cytochrome P450 102A1 (BM3) / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: MHHHHHHIKE MPQPKTFGEL KNLPLLNTDK PVQALMKIAD ELGEIFKFEA PGRVTRYLSS QRLIKEACDE SRFDKNLSQA LKFVRDFFG DGLFTSWTHE KNWKKAHNIL LPSFSQQAMK GYHAMMVDIA VQLVQKWERL NADEHIEVPE DMTRLTLDTI G LCGFNYRF ...String:
MHHHHHHIKE MPQPKTFGEL KNLPLLNTDK PVQALMKIAD ELGEIFKFEA PGRVTRYLSS QRLIKEACDE SRFDKNLSQA LKFVRDFFG DGLFTSWTHE KNWKKAHNIL LPSFSQQAMK GYHAMMVDIA VQLVQKWERL NADEHIEVPE DMTRLTLDTI G LCGFNYRF NSFYRDQPHP FITSMVRALD EAMNKLQRAN PDDPAYDENK RQFQEDIKVM NDLVDKIIAD RKASGEQSDD LL THMLNGK DPETGEPLDD ENIRYQIITF LIAGHETTSG LLSFALYFLV KNPHVLQKAA EEAARVLVDP VPSYKQVKQL KYV GMVLNE ALRLWPTAPA FSLYAKEDTV LGGEYPLEKG DELMVLIPQL HRDKTIWGDD VEEFRPERFE NPSAIPQHAF KPFG NGQRA CIGQQFALHE ATLVLGMMLK HFDFEDHTNY ELDIKETLTL KPEGFVVKAK SKKIPLAENA HNTPLLVLYG SNMGT AEGT ARDLADIAMS KGFAPQVATL DSHAGNLPRE GAVLIVTASY NGHPPDNAKQ FVDWLDQASA DEVKGVRYSV FGCGDK NWA TTYQKVPAFI DETLAAKGAE NIADRGEADA SDDFEGTYEE WREHMWSDVA AYFNLDIENS EDNKSTLSLQ FVDSAAD MP LAKMHGAFST NVVASKELQQ PGSARSTRHL EIELPKEASY QEGDHLGVIP RNYEGIVNRV TARFGLDASQ QIRLEAEE E KLAHLPLAKT VSVEELLQYV ELQDPVTRTQ LRAMAAKTVC PPHKVELEAL LEKQAYKEQV LAKRLTMLEL LEKYPACEM KFSEFIALLP SIRPRYYSIS SSPRVDEKQA SITVSVVSGE AWSGYGEYKG IASNYLAELQ EGDTITCFIS TPQSEFTLPK DPETPLIMV GPGTGVAPFR GFVQARKQLK EQGQSLGEAH LYFGCRSPHE DYLYQEELEN AQSEGIITLH TAFSRMPNQP K TYVQHVME QDGKKLIELL DQGAHFYICG DGSQMAPAVE ATLMKSYADV HQVSEADARL WLQQLEEKGR YAKDVWAG

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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

Concentration3 mg/mL
BufferpH: 7.4 / Details: phosphate-buffered saline
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 0.12 nm / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK I
DetailsThe sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 59611

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