|Entry||Database: PDB / ID: 4uf8|
|Title||Electron cryo-microscopy structure of PB1-p62 filaments|
|Keywords||SIGNALING 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
|Biological species||HOMO SAPIENS (human)|
|Method||ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 10.9 Å|
|Authors||Ciuffa, R. / Lamark, T. / Tarafder, A. / Guesdon, A. / Rybina, S. / Hagen, W.J.H. / Johansen, T. / Sachse, C.|
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
A: SEQUESTOSOME-1x 20
|Symmetry||Helical 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 °)|
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
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction|
|Component||Name: PB1(1-102) DOMAIN OF P62 SQSTM1 / Type: COMPLEX|
|Buffer solution||Name: 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|
|Specimen||Conc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: HOLEY CARBON|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE|
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, TEMPERATURE- 77, INSTRUMENT- HOMEMADE PLUNGER METHOD- BACKSIDE BLOTTING,
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS / Date: Feb 21, 2014|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm|
|Image recording||Electron dose: 15 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)|
|Image scans||Num. digital images: 994|
|Radiation wavelength||Relative weight: 1|
|CTF correction||Details: CTFFIND, CONVOLUTION IMAGES WIENER FILTER RECONSTRUCTION|
|3D reconstruction||Method: 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 building||Protocol: RIGID BODY FIT / Space: REAL / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--NMR|
|Atomic model building||PDB-ID: 2KKC|
|Refinement||Highest resolution: 10.9 Å|
|Refinement step||Cycle: LAST / Highest resolution: 10.9 Å|
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