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- PDB-4uf9: Electron cryo-microscopy structure of PB1-p62 type T filaments -

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Entry
Database: PDB / ID: 4uf9
TitleElectron cryo-microscopy structure of PB1-p62 type T filaments
ComponentsSEQUESTOSOME-1
KeywordsSIGNALING PROTEIN / SELECTIVE AUTOPHAGY / AUTOPHAGY RECEPTOR / AUTOPHAGY SCAFFOLD / P62/SQSTM1 / SINGLE-PARTICLE HELICAL RECONSTRUCTION
Function / homology
Function and homology information


protein localization to perinuclear region of cytoplasm / amphisome / selective autophagy / regulation of Ras protein signal transduction / Lewy body / response to mitochondrial depolarisation / aggrephagy / regulation of protein complex stability / endosome organization / mitophagy ...protein localization to perinuclear region of cytoplasm / amphisome / selective autophagy / regulation of Ras protein signal transduction / Lewy body / response to mitochondrial depolarisation / aggrephagy / regulation of protein complex stability / endosome organization / mitophagy / phagophore assembly site / K63-linked polyubiquitin modification-dependent protein binding / regulation of mitochondrion organization / autolysosome / immune system process / aggresome / sperm midpiece / regulation of I-kappaB kinase/NF-kappaB signaling / autophagy of mitochondrion / ionotropic glutamate receptor binding / autophagosome / sarcomere / inclusion body / response to ischemia / negative regulation of protein ubiquitination / mitochondrion organization / SH2 domain binding / ubiquitin binding / protein kinase C binding / P-body / endosomal transport / protein localization / 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 / intracellular signal transduction / cell differentiation / positive regulation of protein phosphorylation / intracellular membrane-bounded organelle / positive regulation of apoptotic process / protein serine/threonine kinase activity / apoptotic process / ubiquitin protein ligase binding / protein-containing complex binding / protein kinase binding / negative regulation of apoptotic process / negative regulation of transcription by RNA polymerase II / endoplasmic reticulum / enzyme binding / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / cytosol / cytoplasm
PB1 domain / UBA-like superfamily / PB1 domain / Zinc finger, ZZ-type / Zinc finger, ZZ-type superfamily / Sequestosome-1, PB1 domain / Sequestosome-1, UBA domain / Ubiquitin-associated domain
Sequestosome-1
Biological speciesHOMO SAPIENS (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 10.3 Å
AuthorsCiuffa, R. / Lamark, T. / Tarafder, A. / Guesdon, A. / Rybina, S. / Hagen, W.J.H. / Johansen, T. / Sachse, C.
CitationJournal: Cell Rep / Year: 2015
Title: The selective autophagy receptor p62 forms a flexible filamentous helical scaffold.
Authors: Rodolfo Ciuffa / Trond Lamark / Abul K Tarafder / Audrey Guesdon / Sofia Rybina / Wim J H Hagen / Terje Johansen / Carsten Sachse /
Abstract: The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy ...The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMar 15, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 13, 2015Provider: repository / Type: Initial release
Revision 1.1May 27, 2015Group: Database references
Revision 1.2Aug 30, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id

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Structure visualization

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  • Biological unit as representative helical assembly
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Structure viewerMolecule:
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Assembly

Deposited unit
A: SEQUESTOSOME-1
B: SEQUESTOSOME-1
D: SEQUESTOSOME-1


Theoretical massNumber of molelcules
Total (without water)41,1143
Polymers41,1143
Non-polymers00
Water0
1
A: SEQUESTOSOME-1
B: SEQUESTOSOME-1
D: SEQUESTOSOME-1
x 20


Theoretical massNumber of molelcules
Total (without water)822,27760
Polymers822,27760
Non-polymers00
Water0
TypeNameSymmetry operationNumber
helical symmetry operation20
2


  • Idetical with deposited unit in distinct coordinate
  • helical asymmetric unit
TypeNameSymmetry operationNumber
helical symmetry operation1
3


  • Idetical with deposited unit in distinct coordinate
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
transform to helical frame1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 20 / Rise per n subunits: 10.09 Å / Rotation per n subunits: -26.71 °)

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Components

#1: Protein SEQUESTOSOME-1 / EBI3-ASSOCIATED PROTEIN OF 60 KDA / EBIAP / P60 / PHOSPHOTYROSINE-INDEPENDENT LIGAND FOR THE LCK ...EBI3-ASSOCIATED PROTEIN OF 60 KDA / EBIAP / P60 / PHOSPHOTYROSINE-INDEPENDENT LIGAND FOR THE LCK SH2 DOMAIN OF 62 KDA / UBIQUITIN-BINDING PROTEIN P62


Mass: 13704.609 Da / Num. of mol.: 3 / Fragment: PB1 DOMAIN, RESIDUES 1-122
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Plasmid: POPTM-P62-PB1 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 / 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: PB1(1-122) DOMAIN OF P62- SQSTM-1 / Type: COMPLEX
Buffer solutionName: 50 MM TRIS PH 7.5, 100 MM NACL, DTT 4 MM / pH: 7.5 / Details: 50 MM TRIS PH 7.5, 100 MM NACL, DTT 4 MM
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, TEMPERATURE- 77, INSTRUMENT- HOMEMADE PLUNGER, METHOD- BACKSIDE BLOTTING,

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Oct 9, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Image recordingElectron dose: 10 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 443
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2SPRING3D reconstruction
CTF correctionDetails: CTFFIND, CONVOLUTION IMAGES, WIENER FILTER RECONSTRUCTION
3D reconstructionMethod: PROJECTION MATCHING / Resolution: 10.3 Å / Num. of particles: 182238 / Nominal pixel size: 1.372 Å / Actual pixel size: 1.372 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2937. (DEPOSITION ID: 13204).
Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--NMR
Atomic model buildingPDB-ID: 2KKC

2kkc

RefinementHighest resolution: 10.3 Å
Refinement stepCycle: LAST / Highest resolution: 10.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2340 0 0 0 2340

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