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- PDB-6tgy: Cryo-EM structure of p62-PB1 filament (L-type) -

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Basic information

Entry
Database: PDB / ID: 6tgy
TitleCryo-EM structure of p62-PB1 filament (L-type)
ComponentsSequestosome-1
KeywordsAPOPTOSIS / Autophagy
Function / homology
Function and homology information


protein localization to perinuclear region of cytoplasm / selective autophagy / regulation of Ras protein signal transduction / Lewy body / amphisome / response to mitochondrial depolarisation / aggrephagy / regulation of protein complex stability / mitophagy / endosome organization ...protein localization to perinuclear region of cytoplasm / selective autophagy / regulation of Ras protein signal transduction / Lewy body / amphisome / response to mitochondrial depolarisation / aggrephagy / regulation of protein complex stability / mitophagy / endosome organization / phagophore assembly site / K63-linked polyubiquitin modification-dependent protein binding / regulation of mitochondrion organization / autolysosome / immune system process / sperm midpiece / aggresome / regulation of I-kappaB kinase/NF-kappaB signaling / autophagy of mitochondrion / ionotropic glutamate receptor binding / autophagosome / inclusion body / sarcomere / negative regulation of protein ubiquitination / mitochondrion organization / response to ischemia / SH2 domain binding / ubiquitin binding / protein kinase C binding / protein localization / P-body / endosomal transport / positive regulation of long-term synaptic potentiation / positive regulation of protein localization to plasma membrane / macroautophagy / receptor tyrosine kinase binding / PML body / interleukin-1-mediated signaling pathway / autophagy / late endosome / ubiquitin-dependent protein catabolic process / cell differentiation / intracellular signal transduction / positive regulation of protein phosphorylation / positive regulation of apoptotic process / intracellular membrane-bounded organelle / apoptotic process / protein-containing complex binding / protein serine/threonine kinase activity / ubiquitin protein ligase binding / protein kinase binding / negative regulation of apoptotic process / endoplasmic reticulum / negative regulation of transcription by RNA polymerase II / enzyme binding / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / cytosol / cytoplasm
Ubiquitin-associated domain / PB1 domain / Zinc finger, ZZ-type / PB1 domain / Zinc finger, ZZ-type superfamily / Sequestosome-1, PB1 domain / Sequestosome-1, UBA domain / UBA-like superfamily / Phosphatidylinositol 3-kinase Catalytic Subunit; Chain A, domain 1 / Ubiquitin-like (UB roll) ...Ubiquitin-associated domain / PB1 domain / Zinc finger, ZZ-type / PB1 domain / Zinc finger, ZZ-type superfamily / Sequestosome-1, PB1 domain / Sequestosome-1, UBA domain / UBA-like superfamily / Phosphatidylinositol 3-kinase Catalytic Subunit; Chain A, domain 1 / Ubiquitin-like (UB roll) / Roll / Alpha Beta
Sequestosome-1
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsJakobi, A.J. / Huber, S.T. / Mortensen, S.A. / Sachse, C.
Funding support Germany, 3items
OrganizationGrant numberCountry
European CommissionPIEF-GA-2012-331285
European CommissionPCOFUND-GA-2008-229597
German Research FoundationEXC1074 Germany
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis of p62/SQSTM1 helical filaments and their role in cellular cargo uptake.
Authors: Arjen J Jakobi / Stefan T Huber / Simon A Mortensen / Sebastian W Schultz / Anthimi Palara / Tanja Kuhm / Birendra Kumar Shrestha / Trond Lamark / Wim J H Hagen / Matthias Wilmanns / Terje ...Authors: Arjen J Jakobi / Stefan T Huber / Simon A Mortensen / Sebastian W Schultz / Anthimi Palara / Tanja Kuhm / Birendra Kumar Shrestha / Trond Lamark / Wim J H Hagen / Matthias Wilmanns / Terje Johansen / Andreas Brech / Carsten Sachse /
Abstract: p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 ...p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 18, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Sequestosome-1


Theoretical massNumber of molelcules
Total (without water)13,7051
Polymers13,7051
Non-polymers00
Water0
1
A: Sequestosome-1
x 120


Theoretical massNumber of molelcules
Total (without water)1,644,553120
Polymers1,644,553120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation119
MethodUCSF CHIMERA

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Components

#1: Protein Sequestosome-1 / EBI3-associated protein of 60 kDa / p60 / Phosphotyrosine-independent ligand for the Lck SH2 domain ...EBI3-associated protein of 60 kDa / p60 / Phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa / Ubiquitin-binding protein p62


Mass: 13704.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SQSTM1, ORCA, OSIL / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q13501

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: p62-PB1 domain filament (L-type) / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 50 mM TRIS (pH 7.5), 100 mM NaCl, 4 mM DTT
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2277
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1SPRINGparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7PHENIXmodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 77.29 ° / Axial rise/subunit: 4.787 Å / Axial symmetry: C2
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51853 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 2KKC

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