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- EMDB-20785: CYP102A1_A82F_closed -

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Basic information

Entry
Database: EMDB / ID: EMD-20785
TitleCYP102A1_A82F_closed
Map dataEM density map for full-length CYP102A1 in closed state
Sample
  • Complex: Full-length cytochrome P450 CYP102A1 enzyme
    • Protein or peptide: Cytochrome P450 CYP102A1 enzyme
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 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 SciencesGM077430 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
DepositionOct 1, 2019-
Header (metadata) releaseNov 27, 2019-
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: 1.3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20785.png

Downloads & links

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Map

FileDownload / File: emd_20785.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM density map for full-length CYP102A1 in closed state
Voxel sizeX=Y=Z: 3.03 Å
Density
Contour LevelBy AUTHOR: 1.3 / Movie #1: 1.3
Minimum - Maximum-1.4672533 - 6.971842
Average (Standard dev.)0.005221557 (±0.24143578)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 363.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.033.033.03
M x/y/z120120120
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/NS120120120
D min/max/mean-1.4676.9720.005

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Supplemental data

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Sample components

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Entire : Full-length cytochrome P450 CYP102A1 enzyme

EntireName: Full-length cytochrome P450 CYP102A1 enzyme
Components
  • Complex: Full-length cytochrome P450 CYP102A1 enzyme
    • Protein or peptide: Cytochrome P450 CYP102A1 enzyme

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Supramolecule #1: Full-length cytochrome P450 CYP102A1 enzyme

SupramoleculeName: Full-length cytochrome P450 CYP102A1 enzyme / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: recombinant A82F 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 CYP102A1 enzyme

MacromoleculeName: Cytochrome P450 CYP102A1 enzyme / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: NADPH-hemoprotein reductase
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHIKE MPQPKTFGEL KNLPLLNTDK PVQALMKIAD ELGEIFKFEA PGRVTRYLSS QRLIKEACDE SRFDKNLSQA LKFVRDFFGD GLFTSWTHEK NWKKAHNILL PSFSQQAMKG YHAMMVDIAV QLVQKWERLN ADEHIEVPED MTRLTLDTIG LCGFNYRFNS ...String:
MHHHHHHIKE MPQPKTFGEL KNLPLLNTDK PVQALMKIAD ELGEIFKFEA PGRVTRYLSS QRLIKEACDE SRFDKNLSQA LKFVRDFFGD GLFTSWTHEK NWKKAHNILL PSFSQQAMKG YHAMMVDIAV QLVQKWERLN ADEHIEVPED MTRLTLDTIG LCGFNYRFNS FYRDQPHPFI TSMVRALDEA MNKQQRANPD DPAYDENKRQ FQEDIKVMND LVDKIIADRK ASGEQSDDLL THMLNGKDPE TGEPLDDENI RYQIITFLIA GHETTSGLLS FALYFLVKNP HVLQKAAEEA ARVLVDPVPS YKQVKQLKYV GMVLNEALRL WPTAPAFSLY AKEDTVLGGE YPLEKGDELM VLIPQLHRDK TIWGDDVEEF RPERFENPSA IPQHAFKPFG NGQRACIGQQ FALHEATLVL GMMLKHFDFE DHTNYELDIK ETLTLKPEGF VVKAKSKKIP LGGIPSPSTE QSAKKVRKKA ENAHNTPLLV LYGSNMGTAE GTARDLADIA MSKGFAPQVA TLDSHAGNLP REGAVLIVTA SYNGHPPDNA KQFVDWLDQA SADEVKGVRY SVFGCGDKNW ATTYQKVPAF IDETLAAKGA ENIADRGEAD ASDDFEGTYE EWREHMWSDV AAYFNLDIEN SEDNKSTLSL QFVDSAADMP LAKMHGAFST NVVASKELQQ PGSARSTRHL EIELPKEASY QEGDHLGVIP RNYEGIVNRV TARFGLDASQ QIRLEAEEEK LAHLPLAKTV SVEELLQYVE LQDPVTRTQL RAMAAKTVCP PHKVELEALL EKQAYKEQVL AKRLTMLELL EKYPACEMKF SEFIALLPSI RPRYYSISSS PRVDEKQASI TVSVVSGEAW SGYGEYKGIA SNYLAELQEG DTITCFISTP QSEFTLPKDP ETPLIMVGPG TGVAPFRGFV QARKQLKEQG QSLGEAHLYF GCRSPHEDYL YQEELENAQS EGIITLHTAF SRMPNQPKTY VQHVMEQDGK KLIELLDQGA HFYICGDGSQ MAPAVEATLM KSYADVHQVS EADARLWLQQ LEEKGRYAKD VWAG

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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
GridModel: Quantifoil R2/4 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 0.12 nm / Pretreatment - Type: GLOW DISCHARGE
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: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 150000

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