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- EMDB-2937: Electron cryo-microscopy structure of PB1-p62 type T filaments -

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Basic information

Entry
Database: EMDB / ID: EMD-2937
TitleElectron cryo-microscopy structure of PB1-p62 type T filaments
Map data
SamplePB1(1-122) domain of p62/Sqstm1:
Sequestosome-1
KeywordsSelective autophagy / autophagy receptor / autophagy scaffold / p62/SQSTM1 / single-particle helical reconstruction
Function / homology
Function and homology information


response to stress / protein localization to perinuclear region of cytoplasm / protein binding / amphisome / selective autophagy / positive regulation of mitophagy in response to mitochondrial depolarization / regulation of Ras protein signal transduction / Lewy body / protein heterooligomerization / response to mitochondrial depolarisation ...response to stress / protein localization to perinuclear region of cytoplasm / protein binding / amphisome / selective autophagy / positive regulation of mitophagy in response to mitochondrial depolarization / regulation of Ras protein signal transduction / Lewy body / protein heterooligomerization / response to mitochondrial depolarisation / aggrephagy / regulation of autophagy of mitochondrion / 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 / positive regulation of macroautophagy / autophagosome / neurotrophin TRK receptor signaling pathway / sarcomere / inclusion body / response to ischemia / negative regulation of protein ubiquitination / mitochondrion organization / SH2 domain binding / apoptotic signaling pathway / 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 / cytoplasmic vesicle / lysosome / endosome / 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 phosphorylation / protein kinase binding / negative regulation of apoptotic process / negative regulation of transcription by RNA polymerase II / endoplasmic reticulum / enzyme binding / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
PB1 domain / Zinc finger, ZZ-type / UBA-like superfamily / Zinc finger, ZZ-type superfamily / Sequestosome-1, PB1 domain / Sequestosome-1, UBA domain / Ubiquitin-associated domain / PB1 domain / Zinc finger, ZZ-type / Ubiquitin-associated domain / UBA-like superfamily
Sequestosome-1
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 10.3 Å
AuthorsCiuffa R / Lamark T / Tarafder A / Guesdon A / Rybina S / Hagen WJH / 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 ReportSummary, Full report, XML, About validation report
History
DepositionMar 15, 2015-
Header (metadata) releaseApr 1, 2015-
Map releaseMay 20, 2015-
UpdateMay 20, 2015-
Current statusMay 20, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
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  • Surface view colored by cylindrical radius
  • Surface level: 1.5
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  • Surface view with fitted model
  • Atomic models: PDB-4uf9
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4uf9
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4uf9
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2937.map.gz / Format: CCP4 / Size: 18.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.37 Å/pix.
x 290 pix.
= 397.88 Å
1.37 Å/pix.
x 130 pix.
= 178.36 Å
1.37 Å/pix.
x 130 pix.
= 178.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.372 Å
Density
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-2.68769741 - 5.73583364
Average (Standard dev.)-0.00000001 (±0.99999988)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-144
Dimensions130130290
Spacing130130290
CellA: 178.36 Å / B: 178.36 Å / C: 397.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3721.3721.372
M x/y/z130130290
origin x/y/z0.0000.0000.000
length x/y/z178.360178.360397.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-64-64-144
NC/NR/NS130130290
D min/max/mean-2.6885.736-0.000

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Supplemental data

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Sample components

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Entire PB1(1-122) domain of p62/Sqstm1

EntireName: PB1(1-122) domain of p62/Sqstm1 / Details: Helical polymer / Number of components: 1 / Oligomeric State: Helical
MassTheoretical: 13.7 kDa / Experimental: 13.7 kDa / Measured by: Theoretical weight of construct

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Component #1: protein, Sequestosome-1

ProteinName: Sequestosome-1 / a.k.a: p62/SQSTM1 / Oligomeric Details: Helical / Recombinant expression: Yes
MassTheoretical: 13.7 kDa / Experimental: 13.7 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pOPTM-p62-PB1 / Strain: BL21
External referencesInterPro: PB1 domain, UBA-like superfamily, Ubiquitin-associated domain, Zinc finger, ZZ-type
UniProt: Sequestosome-1
Gene Ontology: phagophore assembly site, autophagy of mitochondrion, P-body, P-body, positive regulation of protein phosphorylation, immune system process, protein serine/threonine kinase activity, ...Gene Ontology: phagophore assembly site, autophagy of mitochondrion, P-body, P-body, positive regulation of protein phosphorylation, immune system process, protein serine/threonine kinase activity, protein kinase C binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, protein binding, nucleus, nucleus, nucleoplasm, cytoplasm, cytoplasm, cytoplasm, lysosome, lysosome, endosome, late endosome, autophagosome, autophagosome, autophagosome, endoplasmic reticulum, endoplasmic reticulum, cytosol, cytosol, cytosol, cytosol, cytosol, cytosol, cytosol, cytosol, cytosol, cytosol, protein phosphorylation, ubiquitin-dependent protein catabolic process, autophagy, autophagy, autophagy, apoptotic process, response to stress, protein localization, zinc ion binding, regulation of mitochondrion organization, endosomal transport, inclusion body, aggresome, aggresome, macroautophagy, positive regulation of macroautophagy, PML body, protein kinase binding, cell differentiation, receptor tyrosine kinase binding, cytoplasmic vesicle, intracellular signal transduction, SH2 domain binding, identical protein binding, identical protein binding, identical protein binding, identical protein binding, identical protein binding, protein homodimerization activity, positive regulation of apoptotic process, negative regulation of apoptotic process, regulation of I-kappaB kinase/NF-kappaB signaling, ubiquitin binding, positive regulation of transcription by RNA polymerase II, regulation of Ras protein signal transduction, metal ion binding, neurotrophin TRK receptor signaling pathway, neurotrophin TRK receptor signaling pathway, protein heterooligomerization, extracellular exosome, K63-linked polyubiquitin modification-dependent protein binding, apoptotic signaling pathway, positive regulation of mitophagy in response to mitochondrial depolarization, regulation of autophagy of mitochondrion

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Hand: LEFT HANDED / Delta z: 10.09 Å / Delta phi: 26.71 %deg;
Sample solutionSpecimen conc.: 0.25 mg/mL / Buffer solution: 50 mM Tris pH 7.5, 100 mM NaCl, DTT 4 mM / pH: 7.5
Support filmglow-discharged C-flat 1.2/1.3 and 200 mesh Quantifoil multi-A grids
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 77 K / Method: Backside blotting

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Oct 9, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 10 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 443

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Image processing

ProcessingMethod: helical reconstruction
Details: All of the image processing was carried using the SPRING package.
3D reconstructionAlgorithm: Projection matching / Euler angles: SPIDER / Software: SPRING
CTF correction: CTFFIND, convolution images, Wiener filter reconstruction
Resolution: 10.3 Å / Resolution method: FSC 0.5, semi-independent

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Atomic model buiding

Modeling #1Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL / Details: A homology model was built using
Input PDB model: 2KKC
Output model

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