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- EMDB-3757: CryoEM structure of C. thermophilum fatty acid synthase from nati... -

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Entry
Database: EMDB / ID: EMD-3757
TitleCryoEM structure of C. thermophilum fatty acid synthase from native cell extract
Map data
Sample
  • Complex: The fatty acid synthase complex (FAS1+FAS2, A6B6)
    • Protein or peptide: Fatty acid synthase alpha subunit
    • Protein or peptide: Fatty acid synthase beta subunit
Biological speciesChaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsKastritis PL / O'Reilly F / Bock T / Li Y / Rogon ZM / Buczak K / Romanov N / Betts M / Bui KH / Hagen WJ ...Kastritis PL / O'Reilly F / Bock T / Li Y / Rogon ZM / Buczak K / Romanov N / Betts M / Bui KH / Hagen WJ / Hennrich ML / Mackmull MT / Rappsilber J / Russell R / Bork P / Beck M / Gavin AC
CitationJournal: Mol Syst Biol / Year: 2017
Title: Capturing protein communities by structural proteomics in a thermophilic eukaryote.
Authors: Panagiotis L Kastritis / Francis J O'Reilly / Thomas Bock / Yuanyue Li / Matt Z Rogon / Katarzyna Buczak / Natalie Romanov / Matthew J Betts / Khanh Huy Bui / Wim J Hagen / Marco L Hennrich ...Authors: Panagiotis L Kastritis / Francis J O'Reilly / Thomas Bock / Yuanyue Li / Matt Z Rogon / Katarzyna Buczak / Natalie Romanov / Matthew J Betts / Khanh Huy Bui / Wim J Hagen / Marco L Hennrich / Marie-Therese Mackmull / Juri Rappsilber / Robert B Russell / Peer Bork / Martin Beck / Anne-Claude Gavin /
Abstract: The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their ...The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, , retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for studies. As the components of the captured protein communities are known-at both the protein and complex levels-this study constitutes another step forward toward a molecular understanding of subcellular organization.
History
DepositionJun 7, 2017-
Header (metadata) releaseJul 12, 2017-
Map releaseJul 12, 2017-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3757.png

Downloads & links

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Map

FileDownload / File: emd_3757.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.16 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.13873355 - 0.30307806
Average (Standard dev.)0.0015312372 (±0.014614396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 552.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.162.162.16
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z552.960552.960552.960
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1390.3030.002

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Supplemental data

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Mask #1

Fileemd_3757_msk_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_3757_additional_1.map
Projections & Slices
AxesZYX

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Additional map: CryoEM map of Fatty Acid synthase from C....

Fileemd_3757_additional_2.map
AnnotationCryoEM map of Fatty Acid synthase from C. thermophilum solved from cell extract. Map is unsharpened, unmasked, filtered to FSC=0.143 (RELION).
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Half map: CryoEM half-map 1 of Fatty Acid synthase from...

Fileemd_3757_half_map_1.map
AnnotationCryoEM half-map 1 of Fatty Acid synthase from C. thermophilum solved from cell extract. Map is unsharpened, unmasked, unfiltered.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoEM half-map 2 of Fatty Acid synthase from...

Fileemd_3757_half_map_2.map
AnnotationCryoEM half-map 2 of Fatty Acid synthase from C. thermophilum solved from cell extract. Map is unsharpened, unmasked, unfiltered.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The fatty acid synthase complex (FAS1+FAS2, A6B6)

EntireName: The fatty acid synthase complex (FAS1+FAS2, A6B6)
Components
  • Complex: The fatty acid synthase complex (FAS1+FAS2, A6B6)
    • Protein or peptide: Fatty acid synthase alpha subunit
    • Protein or peptide: Fatty acid synthase beta subunit

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Supramolecule #1: The fatty acid synthase complex (FAS1+FAS2, A6B6)

SupramoleculeName: The fatty acid synthase complex (FAS1+FAS2, A6B6) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
Molecular weightExperimental: 2.6 MDa

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Macromolecule #1: Fatty acid synthase alpha subunit

MacromoleculeName: Fatty acid synthase alpha subunit / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: fatty-acid synthase system
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
SequenceString: MRPEVEQELA HTLLVELLAY QFASPVRWIE TQDVFLAEQM AERIVEIGPA DTLSVMAKRT LASKYEAYD AAKSAQRQIL CYSKDAKEIY YDVDPIEEEP EPAPAQAATP TAPAAPAATP A AAPAPVAA PPPPGVGPAA QVPDAPVTAL EIVRALIAQK LKKPYQEVPL ...String:
MRPEVEQELA HTLLVELLAY QFASPVRWIE TQDVFLAEQM AERIVEIGPA DTLSVMAKRT LASKYEAYD AAKSAQRQIL CYSKDAKEIY YDVDPIEEEP EPAPAQAATP TAPAAPAATP A AAPAPVAA PPPPGVGPAA QVPDAPVTAL EIVRALIAQK LKKPYQEVPL SKAIKDLVGG KS TLQNEIL GDLGKEFGST PEKPEDTPLD ELGAAMQATF DGNLGKTSQG LIARLISSKM PGG FNITTA RKYLETRWGL GPGRQDGVLL LAITMEPPSR LGSEADAKAF LDDVSQKYAA NAGI SLSTA AAAGPAAGAG GGMLMDPAAL EALTSDQKAL FKQQLELIAR YLKLDIRAGD KAYQA SQES AKVLQSQLDL WLAEHGDFYA SGIEPVFSPL KARVYDSSWN WARQDALSMY YDIIFG RLK TVDREIVSQC IRIMNRANPT LLEFMQYHID NCPTDRGETY QLAKELGAQL IENCKEV LN ANPVYKDVAI PTGPKTTIDA RGNLKYEEVP RPSCRKLEHY VQQMAAGGKI SEYGNRIK V QNDLAKIYKL IKQQHKLPKT SQLEIKALYS DIIRALQMNE NQILGQTNGK SLGLPKKGK PKAKTETIPF LHLRKKSVMG WEYNKKLTSL YLDCLEKAAR DGLTFAGKYA LMTGAGAGSI GAEVLQGLI SGGAHVIVTT SRYSREVTEY YQSMYSRYGA RGSQLVVVPF NQGSVQDVNA L VEYIYDTK NGLGWDLDYI VPFAAISEQG RQIDGIDSKS ELAHRIMLTN LIRLLGAVKT QK ASRGYET RPAQVILPLS PNHGTFGSDG LYSESKLGLE TLFNRWESEN WSNYLTICGA IIG WTRGTG LMSGNNIVAE AVEKFGVRTF SQQEMAFNLL GLMAPTIVDL CQNEPVCADL NGGL QFIPN LNELMTRERK NLTETSEIRQ AVTKETAAEN KVVNGEASEA LYKKKIIERR ANIKF DFPP LPDWKKDIQP LNDKLKGMVD LEKVIVVTGF AEVGPWGNSR TRWEMEAYGE FSLEGC IEM AWIMGLIKNY NGLIKGKPYS GWVDAKTGEP VDDKDVKPKY EKYILEHSGI RLIEPEL FG GYDPNKKQLL HEVVIQEDLD PFQCSAETAE QFKREHGDKV EIFEIPESGE YTVRFKKG A TLWIPKALRF DRLVAGQIPT GWDAKRYGIP DDIIQQVDPV CLFVLVSTVE ALLSSGITD PYEFYKYVHV SELGNCIGSG MGGATALRGM HRDRFLDKPL QNDILQESFI NTMSAWVNML LLSSSGPIK TPVAACATAV ESVDVGVETI LEGKARICLV GGFDDFGEEG SYEFANMKAT S NAVDEFAH GRTPQEMSRP TTTTRNGFME SQGSGVQVIM TAKLALEMGV PIYGILALTT TA SDKIGRS VPAPGQGVLT TAREHRGKFP SPLLDINYRR RQIERRTKQV MEEKEAEFEY LAA EIEALK AEGRPQSEIE EYAAHRAAHI EKTAEKQAKE ILRSFGNFFW KNDPTIAPLR GALA VWGLT IDDLDVASFH GTSTKANDKN ESSVICQQLA HLGRKKGNAV LGIFQKYLTG HPKGA AGAW MLNGCLQVLN TGLVPGNRNA DNVDKVMEQF DYIVYPNRSI QTDGIKAFSV TSFGFG QKG AQCIGVHPKY LYATLDEQTY NEYCTKVQAR QKKAYRYFHN GLINNTLFQA KEKAPYT DE QLSAVLLNPD ARVVEDKKTG QLIFPPNFMK LSEKTQAAAQ PKVSLESVLS REARRLES V NTRVGVDVED ISAINTDNDT FLDRNFTEAE QKYCLASKSG RSPQKAFAGR WTAKEAVFK ALGVSSKGAG AALKDIEILV DENGAPTVSL HGAAAEAAKK AGIKSVSVSI SYTDSQAAAI ATAQL

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Macromolecule #2: Fatty acid synthase beta subunit

MacromoleculeName: Fatty acid synthase beta subunit / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO / EC number: fatty-acid synthase system
Source (natural)Organism: Chaetomium thermophilum var. thermophilum DSM 1495 (fungus)
SequenceString: MYGTGTGPQT GAVTPRSSAS LRPLTLSHGS LETSFLIPTG LHFHASRLKD EFIASLPPPT DELAQDDEP SSVPELVARY MGYIANQVAE GEDDAQGSYE EVLKLILNEF ERAFLQGNDV H ALVATLPG IDAKKLEVIR SYFAARAATN RAMRAHQSAL LRAAEEGEAR ...String:
MYGTGTGPQT GAVTPRSSAS LRPLTLSHGS LETSFLIPTG LHFHASRLKD EFIASLPPPT DELAQDDEP SSVPELVARY MGYIANQVAE GEDDAQGSYE EVLKLILNEF ERAFLQGNDV H ALVATLPG IDAKKLEVIR SYFAARAATN RAMRAHQSAL LRAAEEGEAR IYSIFGGQGN IE EYFEELR ELYKTYPSFV GHLIVSSAEL LQILASHPSA EKLYSKGLDI MHWLHNPDAT PDT DYLISA PVSFPLIGLV QLAHYQVTCK VQGLHPGILR DRISGTTGHS QGIVLAAVTA AADS WESFE DLAKSALTIL FWIGARSQQT FPRTSMSPSL LQEAIDNGEG TPTPMLSIRD LPQAE VQKH IDQTNQYLPE DQHISISLIN SPRNLVVSGP PRSLCGLNAQ LRKVKAPTGL DQARIP YSE RKIRFVNRFL PITAPFHSKY LAGAAELIAE DLKDISIEVE RLGIPVYDTN TGEDIRQ TV TGNVVPALIR MITNDPVHWE KATVFPEATH ILDFGPGGIS GLGVLTSRNK DGTGVRVI L AGTVNGTVAE VGYKSELFDR DEEHAVKYAV DWVKEYGPRL IKTSSGRIYV DTKMSRLLG LPPLMVAGMT PTTVPWDFVA ATMNAGYQIE LAGGGYFNAK MMTEAISKIE RAIPPGRGIT VNLIYVNPH AMAWQIPLLG RLRAEGVPIE GLTIGAGVPS IEVANEYIQT LGLKHISFKP G SVDAIQAV INIAKANPTF PVILQWTGGR GGGHHSYEDF HAPILAMYSR IRRQENIILV AG SGFGGAE DTYPYLTGAW STKYGYPPMP FDGCLFGSRM MVAKEAHTSP EAKQAIVDAP GLD DSEWEK TYKGPAGGVI TVRSEMGEPI HKLATRGVLF WAEMDQKIFS LPKEKRVAEL KKNR DYIIR KLNADFQKVW FGKNKKGEVV DLEDMTYGEV VRRMVELLYV KDEKRWIDHS FAKLT ADFI HRVEERFTTA ASQPSLIQSY SDLDEPYSAV ERVLAHYPEA ETQLISAQDV QHFLLL CKR RGQKPVTFVP ALDEDFEFYF KKDSLWQSED LAAVIDRDVG RTCILQGPMA AKHSTKV DE PIKEILDGIH NGHIAALKRD LYDNDESKIP TIEYFGGKLK DPEVQLDFEG VTISYDVH K NTYRVSNNPS VPLPPLDAWL SALAGPNRTW RYALLQSEVI VQGHKYQTNP MKKIFAPAR GLFVEIQYPN DPAKTVITVK EQPRPNRYID VIEAKLVGDK EIVVNLIKDT NALGEPVALP LRFTYRPEA GYAPIHEIME GRNDRIKEFY WRCWFGQDPL DLDAPVTSKF DGGEAVITSE A INEFVHAV GNTGEAFVDR PGKTMYAPMD FAIVVGWKAI TKPIFPRTID GDLLKLVHLS NQ FRMFPGA EPLKKGDKVY TTAQVNAVIN QESGKMVEVC GTITRDGKPV MEVISQFLYR GVY TDYENT FQRKVETPMQ VHLATTKDIA ILRSKQWFVL DDVATPEEFL LGKTLTFRLH TLVH FKNRN VYSHVETRGQ VLVELPTKEI IQVATVEYVA GESHGNPVID YLQRNGQSIE QPVNF ENPI PLGGKAPLQL RAPASNETYA RVSGDYNPIH VSRVFAAYAN LPGTITHGMY SSAAVR SLV ETWAAENKIG RVRSFHASLT GMVLPNDDIN VKLQHVGMVG GRKIIKVEAT NKETEEK VL LGEAEIEQPV TAYVFTGQGS QEQGMGMDLY ANSPVAREVW DRADKYLRDT YGFAITDI V RNNPKELTIH FGGPLGKKIR ANYMAMTFET VAADGSIKSE RIFKDIDENT TSYTFRSPN GLLSATQFTQ PALTLMEKAS FEDMKAKGLV PRDSTFAGHS LGEYSALAAL ADVMPIESLV SVVFYRGLT MQVAVERDAT GRSNYGMCAV NPSRISKTFN EEALRFVVGA VAETTGWLLE I VNYNIANM QYVCAGDLRA LDTLTSVTNF IKAMKIDIEQ MRREYSPDKV KEELVEIIKK CA AETEAKP KPLELQRGFA TIPLRGIDVP FHSTFLRSGV KPFRNFLLKK INKTSIDPAK LIG KYIPNV TAKPFALTKE YFEDVYRLTN SPRIAHVLAN WEKYQDDNST LSASVANTSS ETNG VNGVN GAVDVNGQNG VNGVNGH

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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

Concentration0.04 mg/mL
BufferpH: 7.4
Details: 100 mM HEPES pH 7.4, 95 mM NaCl, 5 mM KCl, 1 mM MgCl2
GridModel: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK II
Details: Blotting and drain time to 3 and 0.5 sec, respectively. Sample volume applied was 3 uL and blot offset was set to -3 mm..
DetailsProtein extract that contains FAS particles as one of the most abundant species

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.5183 µm / Calibrated defocus min: 0.9373 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Digitization - Frames/image: 1-6 / Number grids imaged: 1 / Number real images: 1917 / Average electron dose: 4.0 e/Å2
Details: 4k camera hardware-binned to 2kx2k. Images were collected in movie-mode with 2.8 sec overall exposure. Dose per frame group was 4 e-/A2 for each of the first 6 frames and the last one had a ...Details: 4k camera hardware-binned to 2kx2k. Images were collected in movie-mode with 2.8 sec overall exposure. Dose per frame group was 4 e-/A2 for each of the first 6 frames and the last one had a dose of 24 e-/A2. Overall dose was 48 e-/A2.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7370
Details: The specimen was a native, relatively complex cell extract. FAS particles were sub-selected.
CTF correctionSoftware - Name: CTFFIND (ver. 3.5)
Startup modelType of model: EMDB MAP
EMDB ID:

1623

Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 1.4)
Details: number of single particles: initial picked particles (N): 7370 after 2D classification (N): 4898 after 3D classification (N): Best class had 3933 particles. final refinement (N): 3933
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Details: Pixel size was 2.16 / Number images used: 3933
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient

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