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- EMDB-8072: Alcohol oxidase from Pichia pastoris -

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Basic information

Entry
Database: EMDB / ID: EMD-8072
TitleAlcohol oxidase from Pichia pastoris
Map dataNone
Sample
  • Complex: alcohol oxidase
    • Protein or peptide: Alcohol oxidase 1
  • Ligand: MAGNESIUM ION
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
Keywordsalcohol oxidase peroxisome / oxidoreductase
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
Alcohol oxidase 1 / Alcohol oxidase 1
Similarity search - Component
Biological speciesKomagataella pastoris (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsVonck J / Mills DJ
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, 2016-
Header (metadata) releaseAug 3, 2016-
Map releaseAug 3, 2016-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.052
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.052
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5i68
  • Surface level: 0.052
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5i68
  • Surface level: 0.052
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8072.png

Downloads & links

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Map

FileDownload / File: emd_8072.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 192 pix.
= 218.88 Å		1.14 Å/pix.
x 192 pix.
= 218.88 Å		1.14 Å/pix.
x 192 pix.
= 218.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.052 / Movie #1: 0.052
Minimum - Maximum-0.22297949 - 0.37240782
Average (Standard dev.)-0.00008804758 (±0.022625932)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 218.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.141.141.14
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z218.880218.880218.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-190
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.2230.372-0.000

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Supplemental data

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Sample components

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Entire : alcohol oxidase

EntireName: alcohol oxidase
Components
  • Complex: alcohol oxidase
    • Protein or peptide: Alcohol oxidase 1
  • Ligand: MAGNESIUM ION
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE

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Supramolecule #1: alcohol oxidase

SupramoleculeName: alcohol oxidase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Komagataella pastoris (fungus)
Molecular weightTheoretical: 600 KDa

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Macromolecule #1: Alcohol oxidase 1

MacromoleculeName: Alcohol oxidase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: alcohol oxidase
Source (natural)Organism: Komagataella pastoris (fungus)
Molecular weightTheoretical: 73.992195 KDa
SequenceString: MAIPEEFDIL VLGGGSSGSC IAGRLANLDH SLKVGLIEAG ENNLNNPWVY LPGIYPRNMK LDSKTASFYT SNPSPHLNGR RAIVPCANV LGGGSSINFM MYTRGSASDY DDFQAEGWKT KDLLPLMKKT ETYQRACNNP DIHGFEGPIK VSFGNYTYPV C QDFLRASE ...String:
MAIPEEFDIL VLGGGSSGSC IAGRLANLDH SLKVGLIEAG ENNLNNPWVY LPGIYPRNMK LDSKTASFYT SNPSPHLNGR RAIVPCANV LGGGSSINFM MYTRGSASDY DDFQAEGWKT KDLLPLMKKT ETYQRACNNP DIHGFEGPIK VSFGNYTYPV C QDFLRASE SQGIPYVDDL EDLVTAHGAE HWLKWINRDT GRRSDSAHAF VHSTMRNHDN LYLICNTKVD KIIVEDGRAA AV RTVPSKP LNPKKPSHKI YRARKQIVLS CGTISSPLVL QRSGFGDPIK LRAAGVKPLV NLPGVGRNFQ DHYCFFSPYR IKP QYESFD DFVRGDAEIQ KRVFDQWYAN GTGPLATNGI EAGVKIRPTP EELSQMDESF QEGYREYFED KPDKPVMHYS IIAG FFGDH TKIPPGKYMT MFHFLEYPFS RGSIHITSPD PYAAPDFDPG FMNDERDMAP MVWAYKKSRE TARRMDHFAG EVTSH HPLF PYSSEARALE MDLETSNAYG GPLNLSAGLA HGSWTQPLKK PTAKNEGHVT SNQVELHPDI EYDEEDDKAI ENYIRE HTE TTWHCLGTCS IGPREGSKIV KWGGVLDHRS NVYGVKGLKV GDLSVCPDNV GCNTYTTALL IGEKTATLVG EDLGYSG EA LDMTVPQFKL GTYEKTGLAR F

UniProtKB: Alcohol oxidase 1

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 1 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM

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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

Concentration0.7 mg/mL
BufferpH: 7.5 / Details: Potassium phosphate buffer, 50 mM
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
Details: The grids had been cleaned in chloroform for 2 hrs.
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK I / Details: blot for 11 seconds before plunging.
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeJEOL 3200FSC
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
DetailsData was collected manually
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-21 / Average exposure time: 6.0 sec. / Average electron dose: 51.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.5 µm / Calibrated defocus min: 0.6 µm / Calibrated magnification: 43860 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.2 mm / Nominal magnification: 30000
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER / Cooling holder cryogen: NITROGEN

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Image processing

DetailsThe movie frames were aligned prior to particle picking and the images were binned 3x.
Particle selectionNumber selected: 56544
Startup modelType of model: INSILICO MODEL
In silico model: An initial model was created from negative stain images using EMAN2
Details: D4 symmetry was applied.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Details: RELION was used for the reconstruction / Number images used: 49559
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 1.3) / Details: RELION
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 1.3) / Details: RELION
Final 3D classificationNumber classes: 10 / Avg.num./class: 5600 / Software - Name: RELION (ver. 1.3) / Details: 87.6% of the particles ended up in one 3D class.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

3nne

chain_id: A, source_name: PDB, initial_model_type: experimental model

1gal

chain_id: A, source_name: PDB, initial_model_type: experimental model

3fim

chain_id: B, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 147
Output model

PDB-5i68:
Alcohol oxidase from Pichia pastoris

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