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- PDB-7k1y: PIKfyve/Fig4/Vac14 complex centered on Vac14 - map1 -

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Basic information

Entry
Database: PDB / ID: 7k1y
TitlePIKfyve/Fig4/Vac14 complex centered on Vac14 - map1
ComponentsVac14
KeywordsLIPID BINDING PROTEIN / Lipid kinase / Lipid phosphatase / protein complex
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.25 Å
AuthorsLees, J.A. / Reinisch, K.M. / Li, P.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131715 United States
CitationJournal: Mol Cell / Year: 2020
Title: Insights into Lysosomal PI(3,5)P Homeostasis from a Structural-Biochemical Analysis of the PIKfyve Lipid Kinase Complex.
Authors: Joshua A Lees / PeiQi Li / Nikit Kumar / Lois S Weisman / Karin M Reinisch /
Abstract: The phosphoinositide PI(3,5)P, generated exclusively by the PIKfyve lipid kinase complex, is key for lysosomal biology. Here, we explore how PI(3,5)P levels within cells are regulated. We find the ...The phosphoinositide PI(3,5)P, generated exclusively by the PIKfyve lipid kinase complex, is key for lysosomal biology. Here, we explore how PI(3,5)P levels within cells are regulated. We find the PIKfyve complex comprises five copies of the scaffolding protein Vac14 and one copy each of the lipid kinase PIKfyve, generating PI(3,5)P from PI3P and the lipid phosphatase Fig4, reversing the reaction. Fig4 is active as a lipid phosphatase in the ternary complex, whereas PIKfyve within the complex cannot access membrane-incorporated phosphoinositides due to steric constraints. We find further that the phosphoinositide-directed activities of both PIKfyve and Fig4 are regulated by protein-directed activities within the complex. PIKfyve autophosphorylation represses its lipid kinase activity and stimulates Fig4 lipid phosphatase activity. Further, Fig4 is also a protein phosphatase acting on PIKfyve to stimulate its lipid kinase activity, explaining why catalytically active Fig4 is required for maximal PI(3,5)P production by PIKfyve in vivo.
History
DepositionSep 8, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 21, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 11, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 2, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 6, 2024Group: Database references / Category: database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
B: Vac14
C: Vac14
D: Vac14
E: Vac14
G: Vac14


Theoretical massNumber of molelcules
Total (without water)226,4695
Polymers226,4695
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1210 Å2
ΔGint-18 kcal/mol
Surface area155660 Å2

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Components

#1: Protein
Vac14


Mass: 45293.883 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): Expi293F / Production host: Homo sapiens (human)
Sequence detailsDue to low resolution, only backbone atoms are modeled. The full sequence of the protein is: ...Due to low resolution, only backbone atoms are modeled. The full sequence of the protein is: MNPEKDFAPLTPNIVRALNDKLYEKRKVAALEIEKLVREFVAQNNTVQIKHVIQTLSQEFALSQHPHSRKGGLIGLAACSIALGKDSGLYLKELIEPVLTCFNDADSRLRYYACEALYNIVKVARGAVLPHFNVLFDGLSKLAADPDPNVKSGSELLDRLLKDIVTESNKFDLVSFIPLLRERIYSNNQYARQFIISWILVLESVPDINLLDYLPEILDGLFQILGDNGKEIRKMCEVVLGEFLKEIKKNPSSVKFAEMANILVIHCQTTDDLIQLTAMCWMREFIQLAGRVMLPYSSGILTAVLPCLAYDDRKKSIKEVANVCNQSLMKLVTPEDDELDELRPGQRQAEPTPDDALPKQEGTASGGPDGSCDSSFSSGISVFTAASTERAPVTLHLDGIVQVLNCHLSDTAIGMMTRIAVLKWLYHLYIKTPRKMFRHTDSLFPILLQTLSDESDEVILKDLEVLAEIASSPAGQTDDPGPLDGPDLQASHSELQVPTPGRAGLLNTSGTKGLECSPSTPTMNSYFYKFMINLLKRFSSERKLLEVRGPFIIRQLCLLLNAENIFHSMADILLREEDLKFASTMVHALNTILLTSTELFQLRNQLKDLKTLESQNLFCCLYRSWCHNPVTTVSLCFLTQNYRHAYDLIQKFGDLEVTVDFLAEVDKLVQLIECPIFTYLRLQLLDVKNNPYLIKALYGLLMLLPQSSAFQLLSHRLQCVPNPELLQTEDSLKAAPKSQKADSPSIDYAELLQHFEKVQNKHLEVRHQRSGRGDHLDRRVVL

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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: PIKfyve/Fig4/Vac14 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 4.28 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F
Buffer solutionpH: 7.8
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 / Cs: 2.7 mm
Image recordingElectron dose: 58.4 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 5.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53279 / Symmetry type: POINT

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