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- EMDB-10501: Cryo-EM structure of p62-PB1 filament (L-type) -

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Basic information

Entry
Database: EMDB / ID: EMD-10501
TitleCryo-EM structure of p62-PB1 filament (L-type)
Map dataGlobally sharpened map
Sample
  • Complex: p62-PB1 domain filament (L-type)
    • Protein or peptide: Sequestosome-1
KeywordsAutophagy / apoptosis
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 / response to mitochondrial depolarisation / aggrephagy / negative regulation of toll-like receptor 4 signaling pathway / amphisome / regulation of protein complex stability ...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 / response to mitochondrial depolarisation / aggrephagy / negative regulation of toll-like receptor 4 signaling pathway / amphisome / regulation of protein complex stability / pexophagy / endosome organization / membraneless organelle assembly / molecular sequestering activity / ubiquitin-modified protein reader activity / regulation of mitochondrion organization / phagophore assembly site / regulation of canonical NF-kappaB signal transduction / aggresome / Nuclear events mediated by NFE2L2 / negative regulation of ferroptosis / endosomal transport / autophagy of mitochondrion / intracellular membraneless organelle / temperature homeostasis / K63-linked polyubiquitin modification-dependent protein binding / immune system process / autolysosome / mitophagy / positive regulation of autophagy / signaling adaptor activity / energy homeostasis / inclusion body / sperm midpiece / ionotropic glutamate receptor binding / negative regulation of protein ubiquitination / protein sequestering activity / p75NTR recruits signalling complexes / autophagosome / NF-kB is activated and signals survival / sarcomere / NRIF signals cell death from the nucleus / Pexophagy / SH2 domain binding / PINK1-PRKN Mediated Mitophagy / ubiquitin binding / positive regulation of long-term synaptic potentiation / response to ischemia / positive regulation of protein localization to plasma membrane / molecular condensate scaffold activity / protein kinase C binding / macroautophagy / P-body / protein catabolic process / PML body / receptor tyrosine kinase binding / autophagy / Interleukin-1 signaling / protein import into nucleus / KEAP1-NFE2L2 pathway / late endosome / protein localization / Signaling by ALK fusions and activated point mutants / signaling receptor activity / Neddylation / protein-macromolecule adaptor activity / ubiquitin-dependent protein catabolic process / transcription by RNA polymerase II / cell differentiation / intracellular signal transduction / positive regulation of apoptotic process / positive regulation of protein phosphorylation / intracellular membrane-bounded organelle / ubiquitin protein ligase binding / protein-containing complex binding / apoptotic process / 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 / : / PB1 domain profile. / Ubiquitin associated domain ...Sequestosome-1, UBA domain / Sequestosome-1, PB1 domain / : / UBA 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 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: 3.5 Å
AuthorsHuber ST / Jakobi AJ / Mortensen SA / Sachse C
Funding support Germany, 3 items
OrganizationGrant numberCountry
European CommissionPIEF-GA-2012-331285
European CommissionPCOFUND-GA-2008-229597
German Research FoundationEXC1074 Germany
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-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0325
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0325
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6tgy
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6tgy
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10501.png

Downloads & links

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Map

FileDownload / File: emd_10501.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobally sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 256 pix.
= 266.24 Å		1.04 Å/pix.
x 256 pix.
= 266.24 Å		1.04 Å/pix.
x 256 pix.
= 266.24 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0325 / Movie #1: 0.0325
Minimum - Maximum-0.0596657 - 0.11241907
Average (Standard dev.)0.001132681 (±0.006983475)
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-0.0600.1120.001

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Supplemental data

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Mask #1

Fileemd_10501_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Unfiltered map

Fileemd_10501_additional.map
AnnotationUnfiltered map
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Half map: Unfiltered half map #2

Fileemd_10501_half_map_1.map
AnnotationUnfiltered half map #2
Projections & Slices
AxesZYX

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Histogram

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Half map: Unfiltered half map #1

Fileemd_10501_half_map_2.map
AnnotationUnfiltered half map #1
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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)

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Macromolecule #1: Sequestosome-1

MacromoleculeName: Sequestosome-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / 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

UniProtKB: Sequestosome-1

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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
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 4.787 Å
Applied symmetry - Helical parameters - Δ&Phi: 77.29 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 51853
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2kkc


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-6tgy:
Cryo-EM structure of p62-PB1 filament (L-type)

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