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- PDB-6tgn: Cryo-EM structure of AtNBR1-PB1 filament (L-type) -

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Basic information

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


vacuole / protein polymerization / ubiquitin binding / autophagy / protein transport / zinc ion binding / cytoplasm
Zinc finger, ZZ-type / PB1 domain / UBA-like superfamily / Immunoglobulin-like fold / Next to BRCA1, central domain / PB1 domain
Protein NBR1 homolog
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.9 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 17, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Protein NBR1 homolog


Theoretical massNumber of molelcules
Total (without water)10,1991
Polymers10,1991
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area5770 Å2
MethodPISA

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Components

#1: Protein Protein NBR1 homolog / AtNBR1 / At4g24690


Mass: 10198.557 Da / Num. of mol.: 1
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 (L-type) / Type: COMPLEX / Details: Helical filament of AtNBR 1-PB1 domain (1-95) / Entity ID: 1 / 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

Buffer-ID: 1

IDConc.NameFormula
150 mMHEPES
2150 mMSodium chlorideNaClSodium chloride
30.05 mMTCEP
SpecimenEmbedding 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 / 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)
Image scansMovie frames/image: 7 / Used frames/image: 1-7

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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
9SPRING0.85initial Euler assignment
10SPRING0.85final Euler assignment
11SPRING0.85classification
12SPRING0.853D reconstruction
13PHENIXdev-3500model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -31.44 ° / Axial rise/subunit: 6.721 Å / Axial symmetry: C2
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25387 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
RefinementResolution: 4→49.9 Å / Cor.coef. Fo:Fc: 0.916 / SU B: 46.351 / SU ML: 0.622 / ESU R: 5.547
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.27266 --
Obs0.27266 2714 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 139.102 Å2
Baniso -1Baniso -2Baniso -3
1--0.4 Å2-1.53 Å2-0.76 Å2
2---5.18 Å20.53 Å2
3---5.58 Å2
Refinement stepCycle: 1 / Total: 672
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealDev ideal targetNumber
r_bond_refined_d0.0080.012678
r_bond_other_d
r_angle_refined_deg1.6591.614916
r_angle_other_deg
r_dihedral_angle_1_deg7.877587
r_dihedral_angle_2_deg23.7623.88936
r_dihedral_angle_3_deg13.91315120
r_dihedral_angle_4_deg13.823154
r_chiral_restr0.1020.286
r_gen_planes_refined0.0080.02508
r_gen_planes_other
r_nbd_refined
r_nbd_other
r_nbtor_refined
r_nbtor_other
r_xyhbond_nbd_refined
r_xyhbond_nbd_other
r_metal_ion_refined
r_metal_ion_other
r_symmetry_vdw_refined
r_symmetry_vdw_other
r_symmetry_hbond_refined
r_symmetry_hbond_other
r_symmetry_metal_ion_refined
r_symmetry_metal_ion_other
r_mcbond_it21.76311.168351
r_mcbond_other
r_mcangle_it36.10516.619437
r_mcangle_other
r_scbond_it37.46315.47327
r_scbond_other
r_scangle_it
r_scangle_other
r_long_range_B_refined71.8182358
r_long_range_B_other
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded
LS refinement shellResolution: 4.002→4.105 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.494 210 -
Obs--100 %

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