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- EMDB-12139: Fatty acid synthase (FAS) from Pichia pastoris, including ACP dom... -

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Basic information

Entry
Database: EMDB / ID: EMD-12139
TitleFatty acid synthase (FAS) from Pichia pastoris, including ACP domain resolved by focussed classification
Map dataSharpened map. Derived from focussed classification of ACP domain containing region.
Sample
  • Complex: Fatty acid synthase
    • Protein or peptide: Fatty acid synthase subunit alpha
KeywordsMultienzyme / Complex / Fatty acid / Synthase / BIOSYNTHETIC PROTEIN
Function / homology
Function and homology information


mitotic nuclear membrane biogenesis / palmitic acid biosynthetic process / fatty-acyl-CoA synthase system / fatty-acyl-CoA synthase activity / fatty acid synthase complex / holo-[acyl-carrier-protein] synthase activity / beta-ketoacyl-[acyl-carrier-protein] synthase I / 3-oxoacyl-[acyl-carrier-protein] reductase / 3-oxoacyl-[acyl-carrier-protein] reductase (NADPH) activity / fatty acid synthase activity ...mitotic nuclear membrane biogenesis / palmitic acid biosynthetic process / fatty-acyl-CoA synthase system / fatty-acyl-CoA synthase activity / fatty acid synthase complex / holo-[acyl-carrier-protein] synthase activity / beta-ketoacyl-[acyl-carrier-protein] synthase I / 3-oxoacyl-[acyl-carrier-protein] reductase / 3-oxoacyl-[acyl-carrier-protein] reductase (NADPH) activity / fatty acid synthase activity / 3-oxoacyl-[acyl-carrier-protein] synthase activity / magnesium ion binding
Similarity search - Function
: / Fatty acid synthase subunit alpha, acyl carrier domain / Fatty acid synthase subunit alpha Acyl carrier domain / Fatty acid synthase alpha subunit, yeast / Fatty acid synthase type I, helical / Fatty acid synthase type I helical domain / Holo-[acyl carrier protein] synthase / Phosphopantetheine-protein transferase domain / 4'-phosphopantetheinyl transferase domain / 4'-phosphopantetheinyl transferase domain superfamily ...: / Fatty acid synthase subunit alpha, acyl carrier domain / Fatty acid synthase subunit alpha Acyl carrier domain / Fatty acid synthase alpha subunit, yeast / Fatty acid synthase type I, helical / Fatty acid synthase type I helical domain / Holo-[acyl carrier protein] synthase / Phosphopantetheine-protein transferase domain / 4'-phosphopantetheinyl transferase domain / 4'-phosphopantetheinyl transferase domain superfamily / 4'-phosphopantetheinyl transferase superfamily / Acyl transferase/acyl hydrolase/lysophospholipase / Ketosynthase family 3 (KS3) domain profile. / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / Polyketide synthase, beta-ketoacyl synthase domain / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / Beta-ketoacyl synthase, N-terminal domain / Beta-ketoacyl synthase, C-terminal domain / Thiolase-like / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Fatty acid synthase subunit alpha
Similarity search - Component
Biological speciesKomagataella phaffii GS115 (fungus) / Komagataella phaffii (strain GS115 / ATCC 20864) (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSnowden JS / Alzahrani J
Funding support United Kingdom, Saudi Arabia, 3 items
OrganizationGrant numberCountry
Wellcome Trust102174/B/13/Z United Kingdom
World Health Organization (WHO)2019/883397-O United Kingdom
Saudi Ministry of Education Saudi Arabia
CitationJournal: Sci Rep / Year: 2021
Title: Structural insight into Pichia pastoris fatty acid synthase.
Authors: Joseph S Snowden / Jehad Alzahrani / Lee Sherry / Martin Stacey / David J Rowlands / Neil A Ranson / Nicola J Stonehouse /
Abstract: Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. Serendipitously, we identified FAS as a ...Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. Serendipitously, we identified FAS as a contaminant in a cryoEM dataset of virus-like particles (VLPs) purified from P. pastoris, an important model organism and common expression system used in protein production. From these data, we determined the structure of P. pastoris FAS to 3.1 Å resolution. While the overall organisation of the complex was typical of type I FASs, we identified several differences in both structural and enzymatic domains through comparison with the prototypical yeast FAS from S. cerevisiae. Using focussed classification, we were also able to resolve and model the mobile acyl-carrier protein (ACP) domain, which is key for function. Ultimately, the structure reported here will be a useful resource for further efforts to engineer yeast FAS for synthesis of alternate products.
History
DepositionDec 18, 2020-
Header (metadata) releaseMay 19, 2021-
Map releaseMay 19, 2021-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.021
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bc5
  • Surface level: 0.021
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7bc5
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12139.png

Downloads & links

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Map

FileDownload / File: emd_12139.map.gz / Format: CCP4 / Size: 371.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map. Derived from focussed classification of ACP domain containing region.
Voxel sizeX=Y=Z: 1.065 Å
Density
Contour LevelBy AUTHOR: 0.021 / Movie #1: 0.021
Minimum - Maximum-0.11260153 - 0.21179315
Average (Standard dev.)-0.000025127461 (±0.009525308)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions460460460
Spacing460460460
CellA=B=C: 489.90002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0651.0651.065
M x/y/z460460460
origin x/y/z0.0000.0000.000
length x/y/z489.900489.900489.900
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS460460460
D min/max/mean-0.1130.212-0.000

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Supplemental data

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Mask #1

Fileemd_12139_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map with solvent mask applied.

Fileemd_12139_additional_1.map
AnnotationSharpened map with solvent mask applied.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Focussed mask used for focussed 3D classification of...

Fileemd_12139_additional_2.map
AnnotationFocussed mask used for focussed 3D classification of ACP domain density. The mask only covers the region of the map containing ACP density. This mask was not used for sharpening.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1.

Fileemd_12139_half_map_1.map
AnnotationHalf map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2.

Fileemd_12139_half_map_2.map
AnnotationHalf map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Fatty acid synthase

EntireName: Fatty acid synthase
Components
  • Complex: Fatty acid synthase
    • Protein or peptide: Fatty acid synthase subunit alpha

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Supramolecule #1: Fatty acid synthase

SupramoleculeName: Fatty acid synthase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Komagataella phaffii GS115 (fungus)
Molecular weightTheoretical: 2.6 MDa

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Macromolecule #1: Fatty acid synthase subunit alpha

MacromoleculeName: Fatty acid synthase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: fatty-acyl-CoA synthase system
Source (natural)Organism: Komagataella phaffii (strain GS115 / ATCC 20864) (fungus)
Strain: GS115 / ATCC 20864
Molecular weightTheoretical: 206.375734 KDa
SequenceString: MRPEVEQELS HVLLTELLAY QFASPVRWIE TQDVFLKDYN TERVVEIGPS PTLAGMASRT IKAKYESYDA ALSLQRQVLC YAKDTKEIY YTPDPADIAP PIKEEAETSA AATSSSAPAA AAPVSAAPAA APSGPVAEIP DEPVKAALVL HVLVAHKLKK S LDAVPLSK ...String:
MRPEVEQELS HVLLTELLAY QFASPVRWIE TQDVFLKDYN TERVVEIGPS PTLAGMASRT IKAKYESYDA ALSLQRQVLC YAKDTKEIY YTPDPADIAP PIKEEAETSA AATSSSAPAA AAPVSAAPAA APSGPVAEIP DEPVKAALVL HVLVAHKLKK S LDAVPLSK AIKDLVGGKS TVQNEILGDL GKEFGSTPEK PEDTPLQELA EQFQDTFPGS LGKQTGSLVN RLMSSKMPGG FS LSVARKY LQTRWGLGPG RQDSVLLVAL VNEPGARLSS DGEAKEFLDS CAQKYASGAG ITLAQAAAGG AGSSGAGGAV IDA EAFEEL TKDNRVLARQ QLEVLARYLK YDLTKGEKSL VKEKEASSLL QQELDLWAEE HGEIYAQGIK PVFSHLKART YDSY WNWAR QDALSMYFDI IFGKLTDVDR ETVSQCIQLM NRSNPTLIKF MQYHIDHCPE YKGETYQLAK SLGQQLIDNC IQVAN QDPV YKDISYPTGP HTEVDSKGNI VYKEVNRKSV RKLEQYVFEM SQGGELTKEV QPTIQEDLAK IYEALNKQAS TESQLE FNK LYNSLIEFVE KSKEIEVSKS INAVLASKSS DSDRSAEISS LSEKTSIVDP VSGGIPPETV PFLHLKKKLP SGEWVFD RD TSALFLDGLQ KGAVNGISYK GKNVLITGAG AGSIGAEVLQ GLISGGAKVI VTTSRFSKKV TEYYQDIYAR FGAAGSCL I VVPFNQGSKQ DVEALIDYIY RDVKDEGLGW DLDAVIPFAA IPEAGIEIDE LGSKSELAHR IMLTNLLRLL GEVKKQKFT RAINTRPAQI ILPLSPNHGT FGSDGLYSES KLGLETLFNR WYSESWSEQL TVCGAIIGWT RGTGLMSGNN IIAEGLEKLG VRTFSQKEM AFNILGLMTP ELTEMCQNGP VVADLNGGLQ FIENLREYTA QLRNEIYETS EVRRAVSIET GIETRVVNGE N ADAPYQKA RIEPRANLKF EFPPLKSHKE IQNKAPGLEG LLDLERVIVV TGFGEVSPWG NTRTRWEMEA FGEFSIEGCL EM AWIMGFI KYHNGNLKGK PYTGWIDAKT NEPVEDKDIK KKYEEEILAH AGIRLIEPEL FRGYNPEKKE LIQEVIIEQD MAP FVTDES TAQQYKLQHE DAVDILKSEE SDEYTVTFKK GARLFVPKAL RFDRLVAGQI PTGWDAKRYG ISEDTISQVD PVTL YALVS TIEALLSAGI TDPYEFYKYV HVSEVGNCSG SGMGGVSALR GMFRDRYSDK PVQNDILQES FINTMSAWVN MLLLS SSGP IKTPVGACAT AVESVDIGVE TILSGKAKIC LVGGYDDFQE EGSYEFANMN ATSNSLDEFD HGRTPQEMSR PATTTR NGF MEAQGSGTQV IMNAELAIKM GVPIYAIVAL TATATDKIGR SVPAPGKGIL TTAREHHGSL KTKSPKLDIK YRTRQLN KR KDQIKQWVED ELEYIREEAA ELANSDAKFD AVSFVSERTE EVYREATKQV KMAQQEWGNE FWKNDPRIAP LRGALATF N LTVDDLGVAS FHGTSTKAND KNESITINKM MQHLGRSEGN PVFGVFQKYL TGHPKGAAGA WMLNGAIQIL QTGIVPGNR NADNVDKILE DFEYVLYPSR SIQTDGIKAC SVTSFGFGQK GGQAIVVHPD YLFASLDSET FEEYKTKVEA RYKSTYRYMH NAIIRNTMF VAKSDPPYTD ELEQPVYLDP LARVNNCKKN PSKLVFVNAD VQSKQNFVGK SANDTAKVIS SLTSDVTSGG K GVGVDVEL ISAINNENHT FIERNFTENE ISYCASAPSS KSSLAGTWSA KEAVFKALGV ESKGAGASLK DIEIVRDSKG AP TVVLHGD AKSAASAAGV KNVKVSISHD DVQSVAVAIS EF

UniProtKB: Fatty acid synthase subunit alpha

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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

BufferpH: 7.4 / Details: PBS
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 281 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 60.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: Initial model generated using SGD in RELION 3.0.7.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7)
Details: Asymmetric reconstruction without alignment was performed on each of two subsets (randomly assigned) from an ACP domain density-containing class after focussed 3D classification. This led to ...Details: Asymmetric reconstruction without alignment was performed on each of two subsets (randomly assigned) from an ACP domain density-containing class after focussed 3D classification. This led to the generation of two half-maps, which were used to generate a sharpened map from all the data. Note that the number of particles given above includes particles contributing to multiple symmetrically redundant orientations, as particles were generated through focussed 3D classification.
Number images used: 117012
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.0.7)
Details: After D3 symmetrical reconstruction in RELION, focussed 3D classification was performed on the resultant set of particles and orientations after symmetry expansion (i.e., each particle was ...Details: After D3 symmetrical reconstruction in RELION, focussed 3D classification was performed on the resultant set of particles and orientations after symmetry expansion (i.e., each particle was classified in each symmetrically redundant orientation) with a mask around the ACP domain-containing region of the complex. Classification was performed in RELION 3.0.7, without alignment, and a regularisation parameter ('T' number) of 40. A class containing high-resolution density corresponding to the ACP domain was selected and particles in this class were used for a final reconstruction without alignment.
FSC plot (resolution estimation)

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