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- PDB-6tgp: Cryo-EM structure of AtNBR1-PB1 filament (S-type) -

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Basic information

Entry
Database: PDB / ID: 6tgp
TitleCryo-EM structure of AtNBR1-PB1 filament (S-type)
ComponentsProtein NBR1 homolog
KeywordsSIGNALING PROTEIN / Autophagy / helical filament
Function / homology
Function and homology information


protein targeting to vacuole involved in autophagy / vacuole / protein polymerization / autophagosome / ubiquitin binding / zinc ion binding / cytoplasm
Similarity search - Function
Next to BRCA1, central domain / Ig-like domain from next to BRCA1 gene / PB1 domain / PB1 domain / PB1 domain / 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. ...Next to BRCA1, central domain / Ig-like domain from next to BRCA1 gene / PB1 domain / PB1 domain / PB1 domain / 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. / UBA-like superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Protein NBR1 homolog
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsJakobi, A.J. / Sachse, C.
Funding support Germany, 3items
OrganizationGrant numberCountry
European CommissionPIEF-GA-2012-331285 Germany
European CommissionPCOFUND-GA-2008-229597 Germany
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 17, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 19, 2020Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Protein NBR1 homolog
B: Protein NBR1 homolog


Theoretical massNumber of molelcules
Total (without water)20,3972
Polymers20,3972
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area640 Å2
ΔGint-2 kcal/mol
Surface area10840 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A5 - 92
2010B5 - 92

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Components

#1: Protein Protein NBR1 homolog / AtNBR1 / At4g24690


Mass: 10198.557 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: NBR1, At4g24690, F22K18.110 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9SB64

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: AtNBR1-PB1 (S-type) / Type: COMPLEX / Details: Helical filament of AtNBR1-PB1 domain (1-95) / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
2150 mMSodium chlorideNaClSodium chloride1
30.05 mMTCEP1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 14 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 684

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Processing

SoftwareName: REFMAC / Version: 5.8.0238 / Classification: refinement
EM software
IDNameVersionCategory
1SPRING0.85particle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
7PHENIXdev-3500model fitting
9PHENIXdev-3500model refinement
10SPRING0.85initial Euler assignment
11SPRING0.85final Euler assignment
12SPRING0.85classification
13SPRING0.853D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -31.17 ° / Axial rise/subunit: 5.905 Å / Axial symmetry: C1
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18021 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
RefinementResolution: 4.5→73.46 Å / Cor.coef. Fo:Fc: 0.971 / SU B: 59.134 / SU ML: 0.61
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.28135 --
obs0.28135 5292 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 240.118 Å2
Baniso -1Baniso -2Baniso -3
1--2.75 Å25.65 Å20.11 Å2
2--1.31 Å22.6 Å2
3---1.43 Å2
Refinement stepCycle: 1 / Total: 1344
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0120.0121356
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.6671.6141832
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.8135174
ELECTRON MICROSCOPYr_dihedral_angle_2_deg25.75323.88972
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.43415240
ELECTRON MICROSCOPYr_dihedral_angle_4_deg17.463158
ELECTRON MICROSCOPYr_chiral_restr0.1090.2172
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.021016
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it41.20420.471702
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it69.71830.468874
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it61.51326.724654
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined4741
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Ens-ID: 1 / Number: 4688 / Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Rms dev position: 0.04 Å / Weight position: 0.05

Dom-IDAuth asym-ID
1A
2B
LS refinement shellResolution: 4.5→4.617 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.539 362 -
obs--100 %

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