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- EMDB-2936: Electron cryo-microscopy structure of PB1-p62 filaments -

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Basic information

Entry
Database: EMDB / ID: EMD-2936
TitleElectron cryo-microscopy structure of PB1-p62 filaments
Map data3D reconstruction of PB1(1-102) p62
Sample
  • Sample: PB1(1-102) domain of p62/Sqstm1
  • Protein or peptide: Sequestosome-1
KeywordsSelective autophagy / autophagy receptor / autophagy scaffold / p62/SQSTM1 / single-particle helical reconstruction
Function / homology
Function and homology information


: / brown fat cell proliferation / protein localization to perinuclear region of cytoplasm / protein binding / regulation of Ras protein signal transduction / protein targeting to vacuole involved in autophagy / Lewy body / response to mitochondrial depolarisation / aggrephagy / negative regulation of toll-like receptor 4 signaling pathway ...: / brown fat cell proliferation / protein localization to perinuclear region of cytoplasm / protein binding / regulation of Ras protein signal transduction / protein targeting to vacuole involved in autophagy / Lewy body / response to mitochondrial depolarisation / aggrephagy / negative regulation of toll-like receptor 4 signaling pathway / regulation of autophagy of mitochondrion / amphisome / protein heterooligomerization / regulation of protein complex stability / endosome organization / autophagy of mitochondrion / pexophagy / membraneless organelle assembly / phagophore assembly site / ubiquitin-modified protein reader activity / regulation of mitochondrion organization / regulation of canonical NF-kappaB signal transduction / Nuclear events mediated by NFE2L2 / aggresome / endosomal transport / negative regulation of ferroptosis / intracellular membraneless organelle / K63-linked polyubiquitin modification-dependent protein binding / temperature homeostasis / response to stress / cellular response to stress / neurotrophin TRK receptor signaling pathway / autolysosome / positive regulation of macroautophagy / molecular sequestering activity / immune system process / mitophagy / energy homeostasis / inclusion body / ionotropic glutamate receptor binding / positive regulation of autophagy / signaling adaptor activity / sperm midpiece / negative regulation of protein ubiquitination / p75NTR recruits signalling complexes / SH2 domain binding / NF-kB is activated and signals survival / protein sequestering activity / autophagosome / Pexophagy / NRIF signals cell death from the nucleus / sarcomere / protein kinase C binding / ubiquitin binding / positive regulation of long-term synaptic potentiation / response to ischemia / PINK1-PRKN Mediated Mitophagy / positive regulation of protein localization to plasma membrane / macroautophagy / apoptotic signaling pathway / P-body / molecular condensate scaffold activity / protein catabolic process / PML body / receptor tyrosine kinase binding / autophagy / Interleukin-1 signaling / protein import into nucleus / KEAP1-NFE2L2 pathway / Signaling by ALK fusions and activated point mutants / intracellular protein localization / late endosome / signaling receptor activity / positive regulation of protein phosphorylation / Neddylation / ubiquitin-dependent protein catabolic process / cytoplasmic vesicle / protein-macromolecule adaptor activity / transcription by RNA polymerase II / cell differentiation / lysosome / endosome / intracellular signal transduction / protein phosphorylation / positive regulation of apoptotic process / protein serine/threonine kinase activity / intracellular membrane-bounded organelle / apoptotic process / ubiquitin protein ligase binding / protein-containing complex binding / negative regulation of apoptotic process / protein kinase binding / glutamatergic synapse / enzyme binding / negative regulation of transcription by RNA polymerase II / endoplasmic reticulum / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular exosome
Similarity search - Function
Sequestosome-1, UBA domain / Sequestosome-1, PB1 domain / : / UBA domain / PB1 domain / PB1 domain / PB1 domain / PB1 domain / : / PB1 domain profile. ...Sequestosome-1, UBA domain / Sequestosome-1, PB1 domain / : / UBA domain / PB1 domain / PB1 domain / PB1 domain / PB1 domain / : / PB1 domain profile. / 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 / Zinc finger, ZZ-type superfamily / Zinc finger ZZ-type profile. / Ubiquitin-associated domain / Ubiquitin-associated domain / Ubiquitin-associated domain (UBA) profile. / UBA-like superfamily / UBA-like superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 10.9 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.
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: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4uf8
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4uf8
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4uf8
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2936.png

Downloads & links

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Map

FileDownload / File: emd_2936.map.gz / Format: CCP4 / Size: 4.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of PB1(1-102) p62
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 190 pix.
= 410.4 Å		2.16 Å/pix.
x 80 pix.
= 172.8 Å		2.16 Å/pix.
x 80 pix.
= 172.8 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: 2.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-2.34463453 - 5.80633354
Average (Standard dev.)0.0 (±0.99999958)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-39-39-94
Dimensions8080190
Spacing8080190
CellA: 172.8 Å / B: 172.8 Å / C: 410.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.162.162.16
M x/y/z8080190
origin x/y/z0.0000.0000.000
length x/y/z172.800172.800410.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-39-39-94
NC/NR/NS8080190
D min/max/mean-2.3455.806-0.000

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Supplemental data

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Sample components

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Entire : PB1(1-102) domain of p62/Sqstm1

EntireName: PB1(1-102) domain of p62/Sqstm1
Components
  • Sample: PB1(1-102) domain of p62/Sqstm1
  • Protein or peptide: Sequestosome-1

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Supramolecule #1000: PB1(1-102) domain of p62/Sqstm1

SupramoleculeName: PB1(1-102) domain of p62/Sqstm1 / type: sample / ID: 1000 / Details: Helical polymer / Oligomeric state: Helical / Number unique components: 1
Molecular weightExperimental: 11.3 KDa / Theoretical: 11.3 KDa / Method: Theoretical weight of construct

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Macromolecule #1: Sequestosome-1

MacromoleculeName: Sequestosome-1 / type: protein_or_peptide / ID: 1 / Name.synonym: p62/SQSTM1 / Oligomeric state: Helical / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 11.3 KDa / Theoretical: 11.3 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pOPTM-p62-PB1_K103STOP_E104STOP
SequenceUniProtKB: Sequestosome-1
GO: phagophore assembly site, autophagy of mitochondrion, P-body, P-body, positive regulation of protein phosphorylation, immune system process, protein serine/threonine kinase activity, protein ...GO: 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, intracellular 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 canonical NF-kappaB signal transduction, 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, GO: 0098779, regulation of autophagy of mitochondrion
InterPro: PB1 domain, UBA-like superfamily, Ubiquitin-associated domain, Zinc finger, ZZ-type

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 7.5 / Details: 50 mM Tris pH 7.5, 100 mM NaCl, DTT 4 mM
GridDetails: glow-discharged C-flat 1.2/1.3 and 200 mesh Quantifoil multi-A grids
VitrificationCryogen name: ETHANE / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER / Method: Backside blotting

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateFeb 21, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 994 / Average electron dose: 15 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsAll of the image processing was carried using the SPRING package.
Final reconstructionApplied symmetry - Helical parameters - Δz: 12.99 Å
Applied symmetry - Helical parameters - Δ&Phi: 30.77 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.9 Å / Resolution method: OTHER / Software - Name: SPRING
CTF correctionDetails: CTFFIND, convolution images, Wiener filter reconstruction
Final angle assignmentDetails: SPIDER

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Atomic model buiding 1

Initial modelPDB ID:

2kkc

SoftwareName: Chimera
DetailsA homology model was built using
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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