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- EMDB-10502: Cryo-EM structure of p62-PB1 filament (S-type) -

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Basic information

Entry
Database: EMDB / ID: EMD-10502
TitleCryo-EM structure of p62-PB1 filament (S-type)
Map dataGlobally sharpened map
Sample
  • Complex: p62-PB1 domain filament (L-type)
    • Protein or peptide: Sequestosome-1
Function / homology
Function and homology information


brown fat cell proliferation / protein localization to perinuclear region of cytoplasm / : / protein targeting to vacuole involved in autophagy / regulation of Ras protein signal transduction / Lewy body / aggrephagy / response to mitochondrial depolarisation / amphisome / pexophagy ...brown fat cell proliferation / protein localization to perinuclear region of cytoplasm / : / protein targeting to vacuole involved in autophagy / regulation of Ras protein signal transduction / Lewy body / aggrephagy / response to mitochondrial depolarisation / amphisome / pexophagy / endosome organization / regulation of protein complex stability / autophagy of mitochondrion / phagophore assembly site / regulation of mitochondrion organization / aggresome / regulation of canonical NF-kappaB signal transduction / ubiquitin-modified protein reader activity / K63-linked polyubiquitin modification-dependent protein binding / Nuclear events mediated by NFE2L2 / autolysosome / temperature homeostasis / endosomal transport / immune system process / mitophagy / Signaling by ALK fusions and activated point mutants / autophagosome / signaling adaptor activity / positive regulation of autophagy / energy homeostasis / inclusion body / negative regulation of protein ubiquitination / sperm midpiece / ionotropic glutamate receptor binding / p75NTR recruits signalling complexes / PINK1-PRKN Mediated Mitophagy / Pexophagy / NRIF signals cell death from the nucleus / NF-kB is activated and signals survival / SH2 domain binding / sarcomere / protein kinase C binding / ubiquitin binding / positive regulation of long-term synaptic potentiation / response to ischemia / P-body / positive regulation of protein localization to plasma membrane / macroautophagy / protein catabolic process / protein localization / PML body / receptor tyrosine kinase binding / autophagy / cellular response to reactive oxygen species / Interleukin-1 signaling / protein import into nucleus / KEAP1-NFE2L2 pathway / protein-macromolecule adaptor activity / late endosome / signaling receptor activity / Neddylation / ubiquitin-dependent protein catabolic process / transcription by RNA polymerase II / cell differentiation / intracellular signal transduction / positive regulation of protein phosphorylation / positive regulation of apoptotic process / intracellular membrane-bounded organelle / protein serine/threonine kinase activity / apoptotic process / ubiquitin protein ligase binding / protein-containing complex binding / protein kinase binding / negative regulation of transcription by RNA polymerase II / enzyme binding / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / mitochondrion / extracellular exosome / zinc ion binding / nucleoplasm / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Sequestosome-1, UBA domain / Sequestosome-1, PB1 domain / UBA domain / PB1 domain / PB1 domain / PB1 domain profile. / PB1 domain / Ubiquitin associated domain / Zinc finger ZZ-type signature. / Zinc-binding domain, present in Dystrophin, CREB-binding protein. ...Sequestosome-1, UBA domain / Sequestosome-1, PB1 domain / UBA domain / PB1 domain / PB1 domain / PB1 domain profile. / PB1 domain / 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 superfamily / Zinc finger ZZ-type profile. / Ubiquitin-associated domain / Ubiquitin-associated domain (UBA) profile. / UBA-like superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsHuber ST / Jakobi AJ / Mortensen SA / Sachse C
Funding support Germany, 3 items
OrganizationGrant numberCountry
European CommissionPIEF-GA-2012-331285
German Research FoundationEXC1074 Germany
European CommissionPCOFUND-GA-2008-229597
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis of p62/SQSTM1 helical filaments and their role in cellular cargo uptake.
Authors: Arjen J Jakobi / Stefan T Huber / Simon A Mortensen / Sebastian W Schultz / Anthimi Palara / Tanja Kuhm / Birendra Kumar Shrestha / Trond Lamark / Wim J H Hagen / Matthias Wilmanns / Terje ...Authors: Arjen J Jakobi / Stefan T Huber / Simon A Mortensen / Sebastian W Schultz / Anthimi Palara / Tanja Kuhm / Birendra Kumar Shrestha / Trond Lamark / Wim J H Hagen / Matthias Wilmanns / Terje Johansen / Andreas Brech / Carsten Sachse /
Abstract: p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 ...p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.
History
DepositionNov 18, 2019-
Header (metadata) releaseJan 29, 2020-
Map releaseFeb 12, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0216
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0216
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6th3
  • Surface level: 0.0216
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6th3
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10502.png

Downloads & links

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Map

FileDownload / File: emd_10502.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobally sharpened map
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.0216 / Movie #1: 0.0216
Minimum - Maximum-0.03790701 - 0.088222735
Average (Standard dev.)0.0009815972 (±0.0042340644)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z266.240266.240266.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0380.0880.001

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Supplemental data

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Mask #1

Fileemd_10502_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unfiltered map

Fileemd_10502_additional.map
AnnotationUnfiltered map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map #1

Fileemd_10502_half_map_1.map
AnnotationUnfiltered half map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map #2

Fileemd_10502_half_map_2.map
AnnotationUnfiltered half map #2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : p62-PB1 domain filament (L-type)

EntireName: p62-PB1 domain filament (L-type)
Components
  • Complex: p62-PB1 domain filament (L-type)
    • Protein or peptide: Sequestosome-1

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Supramolecule #1: p62-PB1 domain filament (L-type)

SupramoleculeName: p62-PB1 domain filament (L-type) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Sequestosome-1

MacromoleculeName: Sequestosome-1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.704609 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MASLTVKAYL LGKEDAAREI RRFSFCCSPE PEAEAEAAAG PGPCERLLSR VAALFPALRP GGFQAHYRDE DGDLVAFSSD EELTMAMSY VKDDIFRIYI KEKKECRRDH RPPCAQEAPR NMV

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5 / Details: 50 mM TRIS (pH 7.5), 100 mM NaCl, 4 mM DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 2277 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3)
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.78 Å
Applied symmetry - Helical parameters - Δ&Phi: -26.48 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 51679
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2kkc

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-6th3:
Cryo-EM structure of p62-PB1 filament (S-type)

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