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- EMDB-27988: Yeast VO in complex with Vma12-22p purified via Vma21p-3xFLAG -

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Entry
Database: EMDB / ID: EMD-27988
TitleYeast VO in complex with Vma12-22p purified via Vma21p-3xFLAG
Map data
Sample
  • Complex: Yeast VO in complex with Vma12-22p purified via Vma21p-3xFLAG
    • Protein or peptide: x 11 types
KeywordsV-type / ATPase / assembly / proton / MEMBRANE PROTEIN
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsWang H / Bueler SA / Rubinstein JL
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT166152 Canada
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structural basis of V-ATPase V region assembly by Vma12p, 21p, and 22p.
Authors: Hanlin Wang / Stephanie A Bueler / John L Rubinstein /
Abstract: Vacuolar-type adenosine triphosphatases (V-ATPases) are rotary proton pumps that acidify specific intracellular compartments in almost all eukaryotic cells. These multi-subunit enzymes consist of a ...Vacuolar-type adenosine triphosphatases (V-ATPases) are rotary proton pumps that acidify specific intracellular compartments in almost all eukaryotic cells. These multi-subunit enzymes consist of a soluble catalytic V region and a membrane-embedded proton-translocating V region. V is assembled in the endoplasmic reticulum (ER) membrane, and V is assembled in the cytosol. However, V binds V only after V is transported to the Golgi membrane, thereby preventing acidification of the ER. We isolated V complexes and subcomplexes from bound to V-ATPase assembly factors Vma12p, Vma21p, and Vma22p. Electron cryomicroscopy shows how the Vma12-22p complex recruits subunits a, e, and f to the rotor ring of V while blocking premature binding of V. Vma21p, which contains an ER-retrieval motif, binds the V:Vma12-22p complex, "mature" V, and a complex that appears to contain a ring of loosely packed rotor subunits and the proteins YAR027W and YAR028W. The structures suggest that Vma21p binds assembly intermediates that contain a rotor ring and that activation of proton pumping following assembly of V with V removes Vma21p, allowing V-ATPase to remain in the Golgi. Together, these structures show how Vma12-22p and Vma21p function in V-ATPase assembly and quality control, ensuring the enzyme acidifies only its intended cellular targets.
#1: Journal: Biorxiv / Year: 2022
Title: Structural basis of V-ATPase V0 region assembly by Vma12p, 21p, and 22p
Authors: Wang H / Bueler SA / Rubinstein JL
History
DepositionAug 29, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27988.png

Downloads & links

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Map

FileDownload / File: emd_27988.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-1.0038811 - 2.2426622
Average (Standard dev.)0.0021221787 (±0.103563376)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 329.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27988_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27988_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Yeast VO in complex with Vma12-22p purified via Vma21p-3xFLAG

EntireName: Yeast VO in complex with Vma12-22p purified via Vma21p-3xFLAG
Components
  • Complex: Yeast VO in complex with Vma12-22p purified via Vma21p-3xFLAG
    • Protein or peptide: V-type proton ATPase assembly factor Vma12p
    • Protein or peptide: V-type proton ATPase assembly factor Vma22p
    • Protein or peptide: V-type proton ATPase subunit F
    • Protein or peptide: V-type proton ATPase Voa1p
    • Protein or peptide: V-type proton ATPase subunit a
    • Protein or peptide: V-type proton ATPase subunit c
    • Protein or peptide: V-type proton ATPase subunit c'
    • Protein or peptide: V-type proton ATPase subunit c''
    • Protein or peptide: V-type proton ATPase subunit d
    • Protein or peptide: V-type proton ATPase subunit e
    • Protein or peptide: V-type proton ATPase subunit f

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Supramolecule #1: Yeast VO in complex with Vma12-22p purified via Vma21p-3xFLAG

SupramoleculeName: Yeast VO in complex with Vma12-22p purified via Vma21p-3xFLAG
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: V-type proton ATPase assembly factor Vma12p

MacromoleculeName: V-type proton ATPase assembly factor Vma12p / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MFEIKLNDRI TEFLRKFKNS AKSNEGIDED IDLFLKRHAI PMQSLLFYVK EYRKDSDLQC SIKELLKPLE FEFKPKAVRG LHYSEDFKKK LEFLKYQEQE LEYQSMVKRS KSVFSLQEDD ELTPSQINKQ IKEQVTTVFN VLVSVISVVV AIWYWTGSST NFPVHVRLLL ...String:
MFEIKLNDRI TEFLRKFKNS AKSNEGIDED IDLFLKRHAI PMQSLLFYVK EYRKDSDLQC SIKELLKPLE FEFKPKAVRG LHYSEDFKKK LEFLKYQEQE LEYQSMVKRS KSVFSLQEDD ELTPSQINKQ IKEQVTTVFN VLVSVISVVV AIWYWTGSST NFPVHVRLLL CLFFGILVLV ADVVVYNSYL KKLEEAKVKE KTKVEKKKVL SKITL

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Macromolecule #2: V-type proton ATPase assembly factor Vma22p

MacromoleculeName: V-type proton ATPase assembly factor Vma22p / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MSETRMAQNM DTTDEQYLRL IELLSNYDST LEQLQKGFQD GYIQLSRSNY YNKDSLRGNY GEDYWDETYI GQLMATVEEK NSKVVVEIVK RKAQDKQEKK EEEDNKLTQR KKGTKPEKQK TQSHKLKQDY DPILMFGGVL SVPSSLRQSQ TSFKGCIPLI AQLINYKNEI LTLVETLSEQ E

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Macromolecule #3: V-type proton ATPase subunit F

MacromoleculeName: V-type proton ATPase subunit F / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MAEKRTLIAV IADEDTTTGL LLAGIGQITP ETQEKNFFVY QEGKTTKEEI TDKFNHFTEE RDDIAILLIN QHIAENIRAR VDSFTNAFPA ILEIPSKDHP YDPEKDSVLK RVRKLFGE

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Macromolecule #4: V-type proton ATPase Voa1p

MacromoleculeName: V-type proton ATPase Voa1p / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVFGQLYALF IFTLSCCISK TVQADSSKES SSFISFDKES NWDTISTISS TADVISSVDS AIAVFEFDNF SLLDNLMIDE EYPFFNRFFA NDVSLTVHDD SPLNISQSLS PIMEQFTVDE LPESASDLLY EYSLDDKSIV LFKFTSDAYD LKKLDEFIDS CLSFLEDKSG ...String:
MVFGQLYALF IFTLSCCISK TVQADSSKES SSFISFDKES NWDTISTISS TADVISSVDS AIAVFEFDNF SLLDNLMIDE EYPFFNRFFA NDVSLTVHDD SPLNISQSLS PIMEQFTVDE LPESASDLLY EYSLDDKSIV LFKFTSDAYD LKKLDEFIDS CLSFLEDKSG DNLTVVINSL GWAFEDEDGD DEYATEETLS HHDNNKGKEG DDDILSSIWT EGLLMCLIVS ALLLFILIVA LSWISNLDIT YGALEKSTNP IKKNN

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Macromolecule #5: V-type proton ATPase subunit a

MacromoleculeName: V-type proton ATPase subunit a / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MAEKEEAIFR SAEMALVQFY IPQEISRDSA YTLGQLGLVQ FRDLNSKVRA FQRTFVNEIR RLDNVERQYR YFYSLLKKHD IKLYEGDTDK YLDGSGELYV PPSGSVIDDY VRNASYLEER LIQMEDATDQ IEVQKNDLEQ YRFILQSGDE FFLKGDNTDS TSYMDEDMID ...String:
MAEKEEAIFR SAEMALVQFY IPQEISRDSA YTLGQLGLVQ FRDLNSKVRA FQRTFVNEIR RLDNVERQYR YFYSLLKKHD IKLYEGDTDK YLDGSGELYV PPSGSVIDDY VRNASYLEER LIQMEDATDQ IEVQKNDLEQ YRFILQSGDE FFLKGDNTDS TSYMDEDMID ANGENIAAAI GASVNYVTGV IARDKVATLE QILWRVLRGN LFFKTVEIEQ PVYDVKTREY KHKNAFIVFS HGDLIIKRIR KIAESLDANL YDVDSSNEGR SQQLAKVNKN LSDLYTVLKT TSTTLESELY AIAKELDSWF QDVTREKAIF EILNKSNYDT NRKILIAEGW IPRDELATLQ ARLGEMIARL GIDVPSIIQV LDTNHTPPTF HRTNKFTAGF QSICDCYGIA QYREINAGLP TIVTFPFMFA IMFGDMGHGF LMTLAALSLV LNEKKINKMK RGEIFDMAFT GRYIILLMGV FSMYTGFLYN DIFSKTMTIF KSGWKWPDHW KKGESITATS VGTYPIGLDW AWHGTENALL FSNSYKMKLS ILMGFIHMTY SYFFSLANHL YFNSMIDIIG NFIPGLLFMQ GIFGYLSVCI VYKWAVDWVK DGKPAPGLLN MLINMFLSPG TIDDELYPHQ AKVQVFLLLM ALVCIPWLLL VKPLHFKFTH KKKSHEPLPS TEADASSEDL EAQQLISAMD ADDAEEEEVG SGSHGEDFGD IMIHQVIHTI EFCLNCVSHT ASYLRLWALS LAHAQLSSVL WTMTIQIAFG FRGFVGVFMT VALFAMWFAL TCAVLVLMEG TSAMLHSLRL HWVESMSKFF VGEGLPYEPF AFEYKDMEVA VASASSSASS

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Macromolecule #6: V-type proton ATPase subunit c

MacromoleculeName: V-type proton ATPase subunit c / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MTELCPVYAP FFGAIGCASA IIFTSLGAAY GTAKSGVGIC ATCVLRPDLL FKNIVPVIMA GIIAIYGLVV SVLVCYSLGQ KQALYTGFIQ LGAGLSVGLS GLAAGFAIGI VGDAGVRGSS QQPRLFVGMI LILIFAEVLG LYGLIVALLL NSRATQDVVC

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Macromolecule #7: V-type proton ATPase subunit c'

MacromoleculeName: V-type proton ATPase subunit c' / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MSTQLASNIY APLYAPFFGF AGCAAAMVLS CLGAAIGTAK SGIGIAGIGT FKPELIMKSL IPVVMSGILA IYGLVVAVLI AGNLSPTEDY TLFNGFMHLS CGLCVGFACL SSGYAIGMVG DVGVRKYMHQ PRLFVGIVLI LIFSEVLGLY GMIVALILNT RGSE

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Macromolecule #8: V-type proton ATPase subunit c''

MacromoleculeName: V-type proton ATPase subunit c'' / type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MNKESKDDDM SLGKFSFSHF LYYLVLIVVI VYGLYKLFTG HGSDINFGKF LLRTSPYMWA NLGIALCVGL SVVGAAWGIF ITGSSMIGAG VRAPRITTKN LISIIFCEVV AIYGLIIAIV FSSKLTVATA ENMYSKSNLY TGYSLFWAGI TVGASNLICG IAVGITGATA ...String:
MNKESKDDDM SLGKFSFSHF LYYLVLIVVI VYGLYKLFTG HGSDINFGKF LLRTSPYMWA NLGIALCVGL SVVGAAWGIF ITGSSMIGAG VRAPRITTKN LISIIFCEVV AIYGLIIAIV FSSKLTVATA ENMYSKSNLY TGYSLFWAGI TVGASNLICG IAVGITGATA AISDAADSAL FVKILVIEIF GSILGLLGLI VGLLMAGKAS EFQ

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Macromolecule #9: V-type proton ATPase subunit d

MacromoleculeName: V-type proton ATPase subunit d / type: protein_or_peptide / ID: 9 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MEGVYFNIDN GFIEGVVRGY RNGLLSNNQY INLTQCDTLE DLKLQLSSTD YGNFLSSVSS ESLTTSLIQE YASSKLYHEF NYIRDQSSGS TRKFMDYITY GYMIDNVALM ITGTIHDRDK GEILQRCHPL GWFDTLPTLS VATDLESLYE TVLVDTPLAP YFKNCFDTAE ...String:
MEGVYFNIDN GFIEGVVRGY RNGLLSNNQY INLTQCDTLE DLKLQLSSTD YGNFLSSVSS ESLTTSLIQE YASSKLYHEF NYIRDQSSGS TRKFMDYITY GYMIDNVALM ITGTIHDRDK GEILQRCHPL GWFDTLPTLS VATDLESLYE TVLVDTPLAP YFKNCFDTAE ELDDMNIEII RNKLYKAYLE DFYNFVTEEI PEPAKECMQT LLGFEADRRS INIALNSLQS SDIDPDLKSD LLPNIGKLYP LATFHLAQAQ DFEGVRAALA NVYEYRGFLE TGNLEDHFYQ LEMELCRDAF TQQFAISTVW AWMKSKEQEV RNITWIAECI AQNQRERINN YISVY

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Macromolecule #10: V-type proton ATPase subunit e

MacromoleculeName: V-type proton ATPase subunit e / type: protein_or_peptide / ID: 10 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MSSFYTVVGV FIVVSAMSVL FWIMAPKNNQ AVWRSTVILT LAMMFLMWAI TFLCQLHPLV APRRSDLRPE FAE

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Macromolecule #11: V-type proton ATPase subunit f

MacromoleculeName: V-type proton ATPase subunit f / type: protein_or_peptide / ID: 11 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MRPVVSTGKA WCCTVLSAFG VVILSVIAHL FNTNHESFVG SINDPEDGPA VAHTVYLAAL VYLVFFVFCG FQVYLARRKP SIELR

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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
GridModel: Homemade / Material: COPPER/RHODIUM
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 49.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 17884
FSC plot (resolution estimation)

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