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- PDB-7tap: Cryo-EM structure of archazolid A bound to yeast VO V-ATPase -

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Basic information

Entry
Database: PDB / ID: 7tap
TitleCryo-EM structure of archazolid A bound to yeast VO V-ATPase
Components
  • (V-type proton ATPase subunit ...) x 6
  • V0 assembly protein 1
  • Yeast V-ATPase subunit f
KeywordsMEMBRANE PROTEIN / inhibitor / complex / proton pump / ATPase
Function / homology
Function and homology information


cell wall mannoprotein biosynthetic process / ATPase-coupled ion transmembrane transporter activity / protein localization to vacuolar membrane / 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 / ATPase-coupled ion transmembrane transporter activity / protein localization to vacuolar membrane / 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 / endosomal lumen acidification / vacuolar proton-transporting V-type ATPase, V0 domain / protein targeting to vacuole / vacuole organization / vacuolar proton-transporting V-type ATPase complex / proton-transporting V-type ATPase complex / fungal-type vacuole / vacuolar acidification / cellular hyperosmotic response / fungal-type vacuole membrane / phosphatidylinositol-3,5-bisphosphate binding / proton transmembrane transporter activity / intracellular copper ion homeostasis / Neutrophil degranulation / proton-transporting ATPase activity, rotational mechanism / 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
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 ...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
Archazolid A / 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 speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsKeon, K.A. / Rubinstein, J.L. / Benlekbir, S. / Kirsch, S.H. / Muller, R.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT166152 Canada
CitationJournal: ACS Chem Biol / Year: 2022
Title: Cryo-EM of the Yeast V Complex Reveals Distinct Binding Sites for Macrolide V-ATPase Inhibitors.
Authors: Kristine A Keon / Samir Benlekbir / Susanne H Kirsch / Rolf Müller / John L Rubinstein /
Abstract: Vacuolar-type adenosine triphosphatases (V-ATPases) are proton pumps found in almost all eukaryotic cells. These enzymes consist of a soluble catalytic V region that hydrolyzes ATP and a membrane- ...Vacuolar-type adenosine triphosphatases (V-ATPases) are proton pumps found in almost all eukaryotic cells. These enzymes consist of a soluble catalytic V region that hydrolyzes ATP and a membrane-embedded V region responsible for proton translocation. V-ATPase activity leads to acidification of endosomes, phagosomes, lysosomes, secretory vesicles, and the trans-Golgi network, with extracellular acidification occurring in some specialized cells. Small-molecule inhibitors of V-ATPase have played a crucial role in elucidating numerous aspects of cell biology by blocking acidification of intracellular compartments, while therapeutic use of V-ATPase inhibitors has been proposed for the treatment of cancer, osteoporosis, and some infections. Here, we determine structures of the isolated V complex from bound to two well-known macrolide inhibitors: bafilomycin A1 and archazolid A. The structures reveal different binding sites for the inhibitors on the surface of the proton-carrying c ring, with only a small amount of overlap between the two sites. Binding of both inhibitors is mediated primarily through van der Waals interactions in shallow pockets and suggests that the inhibitors block rotation of the ring. Together, these structures indicate the existence of a large chemical space available for V-ATPase inhibitors that block acidification by binding the c ring.
History
DepositionDec 21, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 23, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 30, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 16, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
D: V-type proton ATPase subunit c'
C: V-type proton ATPase subunit c''
N: V0 assembly protein 1
M: V-type proton ATPase subunit e
E: V-type proton ATPase subunit c
O: Yeast V-ATPase subunit f
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
B: V-type proton ATPase subunit d
A: V-type proton ATPase subunit a, vacuolar isoform
hetero molecules


Theoretical massNumber of molelcules
Total (without water)359,32923
Polymers353,41715
Non-polymers5,9128
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: 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
#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
#4: 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
#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
#7: 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
#8: 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 / 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

#3: 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
#6: 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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Non-polymers , 1 types, 8 molecules

#9: Chemical
ChemComp-KJL / Archazolid A / (1S)-1-{4-[(2S,3S,4E,6E,8S,9S,10R,11E,13Z,15Z,17S,18S,19E,22E)-10,18-dihydroxy-8-methoxy-3,7,9,13,15,17,20,23-octamethyl-24-oxo-1-oxacyclotetracosa-4,6,11,13,15,19,22-heptaen-2-yl]-1,3-thiazol-2-yl}-3-methylbutyl methylcarbamate


Mass: 739.016 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C42H62N2O7S / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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: Cryo-EM structure of archazolid A bound to yeast VO V-ATPase
Type: COMPLEX / Entity ID: #1-#8 / Source: NATURAL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2550 nm / Nominal defocus min: 370 nm
Image recordingElectron dose: 42 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Details: Prototype Falcon 4i camera

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 659182 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00422988
ELECTRON MICROSCOPYf_angle_d0.64631226
ELECTRON MICROSCOPYf_dihedral_angle_d8.0223349
ELECTRON MICROSCOPYf_chiral_restr0.0693711
ELECTRON MICROSCOPYf_plane_restr0.0063851

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