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- PDB-9e7l: Yeast V-ATPase Vo proton channel bound to nanobody 2WVA7 -

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Basic information

Entry
Database: PDB / ID: 9e7l
TitleYeast V-ATPase Vo proton channel bound to nanobody 2WVA7
Components
  • (V-type proton ATPase subunit ...) x 6
  • Nanobody 2WVA7
  • V0 assembly protein 1
  • Yeast V-ATPase subunit f
KeywordsPROTON TRANSPORT / Vacuolar ATPase / Vo proton channel / lipid nanodisc / nanobody
Function / homology
Function and homology information


cell wall mannoprotein biosynthetic process / protein localization to vacuolar membrane / ATPase-coupled ion transmembrane transporter activity / cellular response to alkaline pH / Insulin receptor recycling / Transferrin endocytosis and recycling / polyphosphate metabolic process / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification ...cell wall mannoprotein biosynthetic process / protein localization to vacuolar membrane / ATPase-coupled ion transmembrane transporter activity / cellular response to alkaline pH / Insulin receptor recycling / Transferrin endocytosis and recycling / polyphosphate metabolic process / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / P-type proton-exporting transporter activity / vacuolar transport / vacuolar proton-transporting V-type ATPase, V0 domain / endosomal lumen acidification / vacuole organization / protein targeting to vacuole / proton-transporting V-type ATPase complex / fungal-type vacuole / vacuolar proton-transporting V-type ATPase complex / cellular hyperosmotic response / vacuolar acidification / fungal-type vacuole membrane / phosphatidylinositol-3,5-bisphosphate binding / proton transmembrane transporter activity / proton-transporting ATPase activity, rotational mechanism / intracellular copper ion homeostasis / Neutrophil degranulation / RNA endonuclease activity / proton transmembrane transport / cell periphery / transmembrane transport / endocytosis / ATPase binding / protein-containing complex assembly / intracellular iron ion homeostasis / membrane raft / Golgi membrane / endoplasmic reticulum membrane / membrane
Similarity search - Function
: / V-type proton ATPase subunit f-like / Ribonuclease kappa / V-type proton ATPase subunit S1/VOA1, transmembrane domain / V0 complex accessory subunit Ac45/VOA1 transmembrane domain / ATPase, V0 complex, subunit e1/e2 / ATP synthase subunit H / ATPase, V0 complex, subunit d / V-ATPase proteolipid subunit C, eukaryotic / ATPase, V0 complex, subunit 116kDa, eukaryotic ...: / V-type proton ATPase subunit f-like / Ribonuclease kappa / V-type proton ATPase subunit S1/VOA1, transmembrane domain / V0 complex accessory subunit Ac45/VOA1 transmembrane domain / ATPase, V0 complex, subunit e1/e2 / ATP synthase subunit H / ATPase, V0 complex, subunit d / V-ATPase proteolipid subunit C, eukaryotic / ATPase, V0 complex, subunit 116kDa, eukaryotic / ATPase, V0 complex, c/d subunit / V-type ATPase subunit C/d / V-type ATP synthase subunit c/d subunit superfamily / V-type ATP synthase c/d subunit, domain 3 superfamily / ATP synthase (C/AC39) subunit / V-ATPase proteolipid subunit / V-type ATPase, V0 complex, 116kDa subunit family / V-type ATPase 116kDa subunit family / V-ATPase proteolipid subunit C-like domain / F/V-ATP synthase subunit C superfamily / ATP synthase subunit C
Similarity search - Domain/homology
V-type proton ATPase subunit f / V-type proton ATPase subunit c'' / V-type proton ATPase subunit c / V-type proton ATPase subunit d / V-type proton ATPase subunit a, vacuolar isoform / V-type proton ATPase subunit c' / V0 assembly protein 1 / V-type proton ATPase subunit e
Similarity search - Component
Biological speciesLama glama (llama)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsWilkens, S. / Knight, K. / Roh, S.-H. / Park, J.B.
Funding support United States, Korea, Republic Of, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM141908 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM058600 United States
National Research Foundation (NRF, Korea)2020R1A6C101A183 Korea, Republic Of
National Research Foundation (NRF, Korea)2021M3A9I4021220 Korea, Republic Of
CitationJournal: bioRxiv / Year: 2025
Title: Monoclonal nanobodies alter the activity and assembly of the yeast vacuolar H-ATPase.
Authors: Kassidy Knight / Jun Bae Park / Rebecca A Oot / Md Murad Khan / Soung-Hun Roh / Stephan Wilkens /
Abstract: The vacuolar ATPase (V-ATPase; VV) is a multi-subunit rotary nanomotor proton pump that acidifies organelles in virtually all eukaryotic cells, and extracellular spaces in some specialized tissues of ...The vacuolar ATPase (V-ATPase; VV) is a multi-subunit rotary nanomotor proton pump that acidifies organelles in virtually all eukaryotic cells, and extracellular spaces in some specialized tissues of higher organisms. Evidence suggests that metastatic breast cancers mislocalize V-ATPase to the plasma membrane to promote cell survival and facilitate metastasis, making the V-ATPase a potential drug target. We have generated a library of camelid single-domain antibodies (Nanobodies; Nbs) against lipid-nanodisc reconstituted yeast V-ATPase V proton channel subcomplex. Here, we present an in-depth characterization of three anti-V Nbs using biochemical and biophysical experiments. We find that the Nbs bind V with high affinity, with one Nb inhibiting holoenzyme activity and another one preventing enzyme assembly. Using cryoEM, we find that two of the Nbs bind the subunit ring of the V on the lumen side of the complex. Additionally, we show that one of the Nbs raised against yeast V can pull down human V-ATPase (VV). Our research demonstrates Nb versatility to target and modulate the activity of the V-ATPase, and highlights the potential for future therapeutic Nb development.
History
DepositionNov 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release
Revision 1.0Feb 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 5, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Feb 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 5, 2025Data content type: Mask / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 28, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: V-type proton ATPase subunit d
C: V-type proton ATPase subunit c''
D: V-type proton ATPase subunit c'
X: Nanobody 2WVA7
F: V-type proton ATPase subunit c
G: V-type proton ATPase subunit c
H: V-type proton ATPase subunit c
I: V-type proton ATPase subunit c
J: V-type proton ATPase subunit c
K: V-type proton ATPase subunit c
L: V-type proton ATPase subunit c
M: V-type proton ATPase subunit e
N: V0 assembly protein 1
O: Yeast V-ATPase subunit f
W: Nanobody 2WVA7
V: Nanobody 2WVA7
U: Nanobody 2WVA7
E: V-type proton ATPase subunit c
T: Nanobody 2WVA7
S: Nanobody 2WVA7
R: Nanobody 2WVA7
Q: Nanobody 2WVA7
A: V-type proton ATPase subunit a, vacuolar isoform


Theoretical massNumber of molelcules
Total (without water)472,74823
Polymers472,74823
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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V-type proton ATPase subunit ... , 6 types, 13 molecules BCDFGHIJKLEMA

#1: Protein V-type proton ATPase subunit d / V-ATPase subunit d / V-ATPase 39 kDa subunit / V-ATPase subunit M39 / Vacuolar proton pump subunit d


Mass: 39822.484 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32366
#2: Protein V-type proton ATPase subunit c'' / V-ATPase subunit c'' / V-ATPase 22 kDa proteolipid subunit / Vacuolar proton pump c'' subunit


Mass: 22610.641 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P23968
#3: Protein V-type proton ATPase subunit c' / V-ATPase subunit c' / Proteolipid protein VMA11 / Trifluoperazine resistance protein 3 / V-ATPase ...V-ATPase subunit c' / Proteolipid protein VMA11 / Trifluoperazine resistance protein 3 / V-ATPase 16 kDa proteolipid subunit 2 / Vacuolar proton pump c' subunit


Mass: 17046.361 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32842
#5: Protein
V-type proton ATPase subunit c / V-ATPase subunit c / Guanine nucleotide exchange factor 2 / V-ATPase 16 kDa proteolipid subunit 1 / ...V-ATPase subunit c / Guanine nucleotide exchange factor 2 / V-ATPase 16 kDa proteolipid subunit 1 / Vacuolar proton pump c subunit


Mass: 16357.501 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P25515
#6: Protein V-type proton ATPase subunit e / V-ATPase subunit e / Low dye-binding protein 10 / Vacuolar proton pump subunit e


Mass: 8387.065 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q3E7B6
#9: Protein V-type proton ATPase subunit a, vacuolar isoform / V-ATPase a 1 subunit / V-ATPase 95 kDa subunit / Vacuolar pH protein 1 / Vacuolar proton pump a ...V-ATPase a 1 subunit / V-ATPase 95 kDa subunit / Vacuolar pH protein 1 / Vacuolar proton pump a subunit / Vacuolar proton translocating ATPase subunit a 1


Mass: 95625.484 Da / Num. of mol.: 1 / Mutation: C-terminal calmodulin binding peptide / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32563

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Protein , 2 types, 2 molecules NO

#7: Protein V0 assembly protein 1


Mass: 29694.885 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53262
#8: Protein Yeast V-ATPase subunit f


Mass: 9369.934 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P0C5R9

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Antibody , 1 types, 8 molecules XWVUTSRQ

#4: Antibody
Nanobody 2WVA7


Mass: 14916.397 Da / Num. of mol.: 8 / Mutation: N-terminal pelB leader sequence
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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Details

Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Yeast V-ATPase Vo proton channel bound to nanobody 2WVA7
Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.540 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.4
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Au-flat 1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 39.96 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM software
IDNameVersionCategory
8PHENIXmodel refinement
11RELION5final Euler assignment
13RELION53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53956 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00229776
ELECTRON MICROSCOPYf_angle_d0.47640406
ELECTRON MICROSCOPYf_dihedral_angle_d11.83910427
ELECTRON MICROSCOPYf_chiral_restr0.0364709
ELECTRON MICROSCOPYf_plane_restr0.0035077

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