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- PDB-5i68: Alcohol oxidase from Pichia pastoris -

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Basic information

Entry
Database: PDB / ID: 5i68
TitleAlcohol oxidase from Pichia pastoris
ComponentsAlcohol oxidase 1
KeywordsOXIDOREDUCTASE / alcohol oxidase peroxisome
Function / homology
Function and homology information


methane catabolic process / alcohol oxidase activity / alcohol oxidase / methanol metabolic process / peroxisomal matrix / flavin adenine dinucleotide binding
Similarity search - Function
GMC oxidoreductases signature 1. / Glucose-methanol-choline oxidoreductase / Glucose-methanol-choline oxidoreductase, N-terminal / GMC oxidoreductase / Glucose-methanol-choline oxidoreductase, C-terminal / GMC oxidoreductase / FAD/NAD(P)-binding domain superfamily
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / Alcohol oxidase 1 / Alcohol oxidase 1
Similarity search - Component
Biological speciesKomagataella pastoris (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsVonck, J. / Mills, D.J. / Parcej, D.N.
CitationJournal: PLoS One / Year: 2016
Title: Structure of Alcohol Oxidase from Pichia pastoris by Cryo-Electron Microscopy.
Authors: Janet Vonck / David N Parcej / Deryck J Mills /
Abstract: The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol ...The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes.
History
DepositionFeb 16, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Aug 3, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2017Group: Data collection / Category: em_imaging_optics / em_software
Item: _em_imaging_optics.energyfilter_name / _em_software.name
Revision 1.2Feb 7, 2018Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.energyfilter_lower
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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  • Biological unit as software_defined_assembly
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  • Deposited structure unit
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  • Superimposition on EM map
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Alcohol oxidase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,8023
Polymers73,9921
Non-polymers8102
Water0
1
A: Alcohol oxidase 1
hetero molecules
x 8


Theoretical massNumber of molelcules
Total (without water)598,41624
Polymers591,9388
Non-polymers6,47916
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation7
MethodUCSF CHIMERA
SymmetryPoint symmetry: (Schoenflies symbol: D4 (2x4 fold dihedral))

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Components

#1: Protein Alcohol oxidase 1 / / AOX 1 / Methanol oxidase 1 / MOX 1


Mass: 73992.195 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella pastoris (fungus)
References: UniProt: F2QY27, UniProt: P04842*PLUS, alcohol oxidase
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE / Flavin adenine dinucleotide


Mass: 785.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM

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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: alcohol oxidase / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.5 / Details: Potassium phosphate buffer, 50 mM
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportDetails: The grids had been cleaned in chloroform for 2 hrs.
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 70 % / Chamber temperature: 283 K / Details: blot for 11 seconds before plunging

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Electron microscopy imaging

MicroscopyModel: JEOL 3200FSC / Details: Data was collected manually
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 30000 X / Calibrated magnification: 43860 X / Calibrated defocus min: 600 nm / Calibrated defocus max: 2500 nm / Cs: 4.2 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL 3200FSC CRYOHOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 51 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansSampling size: 5 µm / Movie frames/image: 30 / Used frames/image: 2-21

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Processing

EM software
IDNameVersionCategoryDetails
1EMANparticle selectionEMAN boxer was used semi-automatically
4RELION1.3CTF correction
7UCSF Chimeramodel fittingChimera was used to fit homologous parts of three structures to the map.
9RELION1.3initial Euler assignment
10RELION1.3final Euler assignment
11RELION1.3classification
12RELION1.33D reconstruction
13Cootmodel refinementCoot was used to fit the structure based on the homologues and to build unique sequence ab initio
14PHENIXmodel refinementPhenix was used for real-space refinement
Image processingDetails: The movie frames were aligned prior to particle picking and the images were binned 3x.
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 1 Å / B: 1 Å / C: 1 Å / Space group name: 1 / Space group num: 1
CTF correctionDetails: CTF was determined by CTFFIND3 inside the RELION software
Type: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 56544
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49559 / Details: RELION was used for the reconstruction / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 147 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
13NNE

3nne

A1
21GAL

1gal

A1
33FIM

3fim

B1

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