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- PDB-8f6t: Cryo-EM structure of alkane 1-monooxygenase AlkB-AlkG complex fro... -

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Basic information

Entry
Database: PDB / ID: 8f6t
TitleCryo-EM structure of alkane 1-monooxygenase AlkB-AlkG complex from Fontimonas thermophila
ComponentsAlkane 1-monooxygenase
KeywordsOXIDOREDUCTASE / Electron transfer complex / iron-sulfur cluster / histidine-diiron center / hydrophobic alkane binding pocket
Function / homology
Function and homology information


monooxygenase activity / lipid metabolic process / iron ion binding / plasma membrane
Similarity search - Function
Alkane/xylene monooxygenase / Fatty acid desaturase domain / Fatty acid desaturase / Rubredoxin, iron-binding site / Rubredoxin signature. / Rubredoxin domain / Rubredoxin / Rubredoxin-like domain / Rubredoxin-like domain profile.
Similarity search - Domain/homology
DODECANE / : / Alkane 1-monooxygenase
Similarity search - Component
Biological speciesFontimonas thermophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.76 Å
AuthorsChai, J. / Guo, G. / McSweeney, S. / Shanklin, J. / Liu, Q.
Funding support United States, 1items
OrganizationGrant numberCountry
Department of Energy (DOE, United States) United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural basis for enzymatic terminal C-H bond functionalization of alkanes.
Authors: Jin Chai / Gongrui Guo / Sean M McSweeney / John Shanklin / Qun Liu /
Abstract: Alkane monooxygenase (AlkB) is a widely occurring integral membrane metalloenzyme that catalyzes the initial step in the functionalization of recalcitrant alkanes with high terminal selectivity. AlkB ...Alkane monooxygenase (AlkB) is a widely occurring integral membrane metalloenzyme that catalyzes the initial step in the functionalization of recalcitrant alkanes with high terminal selectivity. AlkB enables diverse microorganisms to use alkanes as their sole carbon and energy source. Here we present the 48.6-kDa cryo-electron microscopy structure of a natural fusion from Fontimonas thermophila between AlkB and its electron donor AlkG at 2.76 Å resolution. The AlkB portion contains six transmembrane helices with an alkane entry tunnel within its transmembrane domain. A dodecane substrate is oriented by hydrophobic tunnel-lining residues to present a terminal C-H bond toward a diiron active site. AlkG, an [Fe-4S] rubredoxin, docks via electrostatic interactions and sequentially transfers electrons to the diiron center. The archetypal structural complex presented reveals the basis for terminal C-H selectivity and functionalization within this broadly distributed evolutionary class of enzymes.
History
DepositionNov 17, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 26, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3May 22, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Alkane 1-monooxygenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,2805
Polymers52,9421
Non-polymers3384
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Alkane 1-monooxygenase


Mass: 52942.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Fontimonas thermophila (bacteria) / Gene: SAMN04488120_10311 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1I2I8Z9, alkane 1-monooxygenase
#2: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-D12 / DODECANE


Mass: 170.335 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H26 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Electron transfer complex of AlkB and AlkG / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Fontimonas thermophila (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 700 nm
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46953 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 104.93 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413585
ELECTRON MICROSCOPYf_angle_d0.57354898
ELECTRON MICROSCOPYf_chiral_restr0.041516
ELECTRON MICROSCOPYf_plane_restr0.0046624
ELECTRON MICROSCOPYf_dihedral_angle_d9.9053485

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