+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8072 | |||||||||
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Title | Alcohol oxidase from Pichia pastoris | |||||||||
Map data | None | |||||||||
Sample |
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Keywords | alcohol 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 | |||||||||
Biological species | Komagataella pastoris (fungus) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.37 Å | |||||||||
Authors | Vonck J / Mills DJ | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_8072.map.gz | 25 MB | EMDB map data format | |
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Header (meta data) | emd-8072-v30.xml emd-8072.xml | 15.3 KB 15.3 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_8072_fsc.xml | 6.8 KB | Display | FSC data file |
Images | emd_8072.png | 259.2 KB | ||
Filedesc metadata | emd-8072.cif.gz | 6.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8072 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8072 | HTTPS FTP |
-Validation report
Summary document | emd_8072_validation.pdf.gz | 316.5 KB | Display | EMDB validaton report |
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Full document | emd_8072_full_validation.pdf.gz | 315.6 KB | Display | |
Data in XML | emd_8072_validation.xml.gz | 9.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8072 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8072 | HTTPS FTP |
-Related structure data
Related structure data | 5i68MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_8072.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | None | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.14 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : alcohol oxidase
Entire | Name: alcohol oxidase |
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Components |
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-Supramolecule #1: alcohol oxidase
Supramolecule | Name: alcohol oxidase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Komagataella pastoris (fungus) |
Molecular weight | Theoretical: 600 KDa |
-Macromolecule #1: Alcohol oxidase 1
Macromolecule | Name: Alcohol oxidase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: alcohol oxidase |
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Source (natural) | Organism: Komagataella pastoris (fungus) |
Molecular weight | Theoretical: 73.992195 KDa |
Sequence | String: 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 |
-Macromolecule #2: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #3: FLAVIN-ADENINE DINUCLEOTIDE
Macromolecule | Name: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 1 / Formula: FAD |
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Molecular weight | Theoretical: 785.55 Da |
Chemical component information | ChemComp-FAD: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.7 mg/mL |
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Buffer | pH: 7.5 / Details: Potassium phosphate buffer, 50 mM |
Grid | Model: 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. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK I / Details: blot for 11 seconds before plunging. |
Details | This sample was monodisperse |
-Electron microscopy
Microscope | JEOL 3200FSC |
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Specialist optics | Energy filter - Name: In-column Omega Filter / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV |
Details | Data was collected manually |
Image recording | Film 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 beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated 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 stage | Specimen holder model: JEOL 3200FSC CRYOHOLDER / Cooling holder cryogen: NITROGEN |
+Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: AB INITIO MODEL / Overall B value: 147 | ||||||||
Output model | PDB-5i68: |