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- PDB-6th3: Cryo-EM structure of p62-PB1 filament (S-type) -

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Basic information

Entry
Database: PDB / ID: 6th3
TitleCryo-EM structure of p62-PB1 filament (S-type)
ComponentsSequestosome-1
KeywordsCYTOSOLIC PROTEIN / Autophagy
Function / homology
Function and homology information


brown fat cell proliferation / protein localization to perinuclear region of cytoplasm / : / protein targeting to vacuole involved in autophagy / regulation of Ras protein signal transduction / Lewy body / aggrephagy / response to mitochondrial depolarisation / amphisome / pexophagy ...brown fat cell proliferation / protein localization to perinuclear region of cytoplasm / : / protein targeting to vacuole involved in autophagy / regulation of Ras protein signal transduction / Lewy body / aggrephagy / response to mitochondrial depolarisation / amphisome / pexophagy / endosome organization / regulation of protein complex stability / autophagy of mitochondrion / phagophore assembly site / aggresome / regulation of mitochondrion organization / regulation of canonical NF-kappaB signal transduction / ubiquitin-modified protein reader activity / K63-linked polyubiquitin modification-dependent protein binding / Nuclear events mediated by NFE2L2 / autolysosome / temperature homeostasis / endosomal transport / immune system process / mitophagy / Signaling by ALK fusions and activated point mutants / autophagosome / signaling adaptor activity / positive regulation of autophagy / energy homeostasis / inclusion body / negative regulation of protein ubiquitination / sperm midpiece / ionotropic glutamate receptor binding / p75NTR recruits signalling complexes / PINK1-PRKN Mediated Mitophagy / Pexophagy / NRIF signals cell death from the nucleus / NF-kB is activated and signals survival / SH2 domain binding / sarcomere / protein kinase C binding / ubiquitin binding / positive regulation of long-term synaptic potentiation / response to ischemia / P-body / positive regulation of protein localization to plasma membrane / macroautophagy / protein catabolic process / protein localization / PML body / receptor tyrosine kinase binding / autophagy / cellular response to reactive oxygen species / Interleukin-1 signaling / protein import into nucleus / KEAP1-NFE2L2 pathway / protein-macromolecule adaptor activity / late endosome / signaling receptor activity / Neddylation / ubiquitin-dependent protein catabolic process / transcription by RNA polymerase II / cell differentiation / intracellular signal transduction / positive regulation of protein phosphorylation / positive regulation of apoptotic process / intracellular membrane-bounded organelle / protein serine/threonine kinase activity / apoptotic process / ubiquitin protein ligase binding / protein-containing complex binding / protein kinase binding / negative regulation of transcription by RNA polymerase II / enzyme binding / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Sequestosome-1, UBA domain / Sequestosome-1, PB1 domain / UBA domain / PB1 domain / PB1 domain / PB1 domain profile. / PB1 domain / Ubiquitin associated domain / Zinc finger ZZ-type signature. / Zinc-binding domain, present in Dystrophin, CREB-binding protein. ...Sequestosome-1, UBA domain / Sequestosome-1, PB1 domain / UBA domain / PB1 domain / PB1 domain / PB1 domain profile. / PB1 domain / Ubiquitin associated domain / Zinc finger ZZ-type signature. / Zinc-binding domain, present in Dystrophin, CREB-binding protein. / Zinc finger, ZZ type / Zinc finger, ZZ-type / Zinc finger, ZZ-type superfamily / Zinc finger ZZ-type profile. / Ubiquitin-associated domain / Ubiquitin-associated domain (UBA) profile. / UBA-like superfamily / Phosphatidylinositol 3-kinase Catalytic Subunit; Chain A, domain 1 / Ubiquitin-like (UB roll) / Roll / Alpha Beta
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4 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.
History
DepositionNov 18, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Sequestosome-1
B: Sequestosome-1
C: Sequestosome-1


Theoretical massNumber of molelcules
Total (without water)41,1143
Polymers41,1143
Non-polymers00
Water0
1
A: Sequestosome-1
B: Sequestosome-1
C: Sequestosome-1
x 25


Theoretical massNumber of molelcules
Total (without water)1,027,84675
Polymers1,027,84675
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
point symmetry operation24
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.: 3
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: all / 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
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 / Specimen holder 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
9PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -26.48 ° / Axial rise/subunit: 9.78 Å / Axial symmetry: C1
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51679 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 2KKC

2kkc

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