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- PDB-4uf8: Electron cryo-microscopy structure of PB1-p62 filaments -

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Basic information

Entry
Database: PDB / ID: 4uf8
TitleElectron cryo-microscopy structure of PB1-p62 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 / sperm midpiece / immune system process / 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 / response to ischemia / mitochondrion organization / SH2 domain binding / ubiquitin binding / protein kinase C binding / P-body / protein localization / 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 / intracellular signal transduction / cell differentiation / 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
PB1 domain / Zinc finger, ZZ-type superfamily / PB1 domain / UBA-like superfamily / Ubiquitin-associated domain / Sequestosome-1, UBA domain / Sequestosome-1, PB1 domain / Zinc finger, ZZ-type
Sequestosome-1
Biological speciesHOMO SAPIENS (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 10.9 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 23, 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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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: SEQUESTOSOME-1
B: SEQUESTOSOME-1
C: SEQUESTOSOME-1
I: SEQUESTOSOME-1


Theoretical massNumber of molelcules
Total (without water)44,4824
Polymers44,4824
Non-polymers00
Water0
1
A: SEQUESTOSOME-1
B: SEQUESTOSOME-1
C: SEQUESTOSOME-1
I: SEQUESTOSOME-1
x 20


Theoretical massNumber of molelcules
Total (without water)889,64680
Polymers889,64680
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: 12.99 Å / Rotation per n subunits: -30.77 °)

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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: 11120.575 Da / Num. of mol.: 4 / Fragment: PB1 DOMAIN, RESIDUES 3-102
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Plasmid: POPTM-P62-PB1_K103STOP_E104STOP / 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(1-102) DOMAIN OF P62 SQSTM1 / 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: Feb 21, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 15 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)
Image scansNum. digital images: 994
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: 10.9 Å / Num. of particles: 50620 / Nominal pixel size: 2.16 Å / Actual pixel size: 2.16 Å
Details: SINGLE-PARTICLE BASED HELICAL RECONSTRUCTION USING SPRING. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2936. (DEPOSITION ID: 13197)
Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--NMR
Atomic model buildingPDB-ID: 2KKC
RefinementHighest resolution: 10.9 Å
Refinement stepCycle: LAST / Highest resolution: 10.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3120 0 0 0 3120

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