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- EMDB-30652: Cryo-EM structure of the Ape4 and Nbr1 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-30652
TitleCryo-EM structure of the Ape4 and Nbr1 complex
Map data
Sample
  • Complex: Ape4 and Nbr1 fusion protein
    • Protein or peptide: Aspartyl aminopeptidase 1,ZZ-type zinc finger-containing protein P35G2.11c,Maltose/maltodextrin-binding periplasmic protein
  • Ligand: ZINC ION
  • Ligand: water
Keywordsaspartyl aminopeptidase / signaling protein / HYDROLASE / autophagy
Function / homology
Function and homology information


NVT complex / aspartyl aminopeptidase / fungal-type vacuole lumen / cytoplasm to vacuole targeting by the NVT pathway / fungal-type vacuole / cargo receptor activity / detection of maltose stimulus / maltose transport complex / metalloaminopeptidase activity / carbohydrate transport ...NVT complex / aspartyl aminopeptidase / fungal-type vacuole lumen / cytoplasm to vacuole targeting by the NVT pathway / fungal-type vacuole / cargo receptor activity / detection of maltose stimulus / maltose transport complex / metalloaminopeptidase activity / carbohydrate transport / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / aminopeptidase activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / multivesicular body / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / metallopeptidase activity / protein folding / outer membrane-bounded periplasmic space / periplasmic space / DNA damage response / Golgi apparatus / proteolysis / zinc ion binding / membrane / cytoplasm / cytosol
Similarity search - Function
Next to BRCA1, central domain / Ig-like domain from next to BRCA1 gene / Peptidase M18 / Peptidase M18, domain 2 / Aminopeptidase I zinc metalloprotease (M18) / 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 ...Next to BRCA1, central domain / Ig-like domain from next to BRCA1 gene / Peptidase M18 / Peptidase M18, domain 2 / Aminopeptidase I zinc metalloprotease (M18) / 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. / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / Immunoglobulin-like fold
Similarity search - Domain/homology
Aspartyl aminopeptidase 1 / Maltose/maltodextrin-binding periplasmic protein / Autophagy receptor Nbr1
Similarity search - Component
Biological speciesSchizosaccharomyces pombe 972h- (yeast) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.26 Å
AuthorsZhang J / Ye K
Funding support China, 3 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32071199, 91940302 China
Chinese Academy of SciencesXDB37010201 China
National Basic Research Program of China (973 Program)2017YFA0504600 China
CitationJournal: EMBO J / Year: 2021
Title: Molecular and structural mechanisms of ZZ domain-mediated cargo selection by Nbr1.
Authors: Ying-Ying Wang / Jianxiu Zhang / Xiao-Man Liu / Yulu Li / Jianhua Sui / Meng-Qiu Dong / Keqiong Ye / Li-Lin Du /
Abstract: In selective autophagy, cargo selectivity is determined by autophagy receptors. However, it remains scarcely understood how autophagy receptors recognize specific protein cargos. In the fission yeast ...In selective autophagy, cargo selectivity is determined by autophagy receptors. However, it remains scarcely understood how autophagy receptors recognize specific protein cargos. In the fission yeast Schizosaccharomyces pombe, a selective autophagy pathway termed Nbr1-mediated vacuolar targeting (NVT) employs Nbr1, an autophagy receptor conserved across eukaryotes including humans, to target cytosolic hydrolases into the vacuole. Here, we identify two new NVT cargos, the mannosidase Ams1 and the aminopeptidase Ape4, that bind competitively to the first ZZ domain of Nbr1 (Nbr1-ZZ1). High-resolution cryo-EM analyses reveal how a single ZZ domain recognizes two distinct protein cargos. Nbr1-ZZ1 not only recognizes the N-termini of cargos via a conserved acidic pocket, similar to other characterized ZZ domains, but also engages additional parts of cargos in a cargo-specific manner. Our findings unveil a single-domain bispecific mechanism of autophagy cargo recognition, elucidate its underlying structural basis, and expand the understanding of ZZ domain-mediated protein-protein interactions.
History
DepositionOct 28, 2020-
Header (metadata) releaseJul 14, 2021-
Map releaseJul 14, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 19
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 19
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7dde
  • Surface level: 12
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7dde
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30652.png

Downloads & links

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Map

FileDownload / File: emd_30652.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 320 pix.
= 332.8 Å		1.04 Å/pix.
x 320 pix.
= 332.8 Å		1.04 Å/pix.
x 320 pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.5 / Movie #1: 19
Minimum - Maximum-16.670127999999998 - 41.296306999999999
Average (Standard dev.)0.05520786 (±1.6046865)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 332.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-16.67041.2960.055

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Supplemental data

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Additional map: #2

Fileemd_30652_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_30652_additional_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ape4 and Nbr1 fusion protein

EntireName: Ape4 and Nbr1 fusion protein
Components
  • Complex: Ape4 and Nbr1 fusion protein
    • Protein or peptide: Aspartyl aminopeptidase 1,ZZ-type zinc finger-containing protein P35G2.11c,Maltose/maltodextrin-binding periplasmic protein
  • Ligand: ZINC ION
  • Ligand: water

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Supramolecule #1: Ape4 and Nbr1 fusion protein

SupramoleculeName: Ape4 and Nbr1 fusion protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: The fusion protein comprises of the full-length Ape4, a linker sequence (GFKKASSSDNKEQ), residues 53-129 of Nbr1, and maltose binding protein (MBP).
Source (natural)Organism: Schizosaccharomyces pombe 972h- (yeast)
Molecular weightTheoretical: 750 KDa

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Macromolecule #1: Aspartyl aminopeptidase 1,ZZ-type zinc finger-containing protein ...

MacromoleculeName: Aspartyl aminopeptidase 1,ZZ-type zinc finger-containing protein P35G2.11c,Maltose/maltodextrin-binding periplasmic protein
type: protein_or_peptide / ID: 1
Details: The fusion protein comprises of the full-length Ape4, a linker sequence (GFKKASSSDNKEQ), residues 53-129 of Nbr1, and maltose binding protein (MBP).
Number of copies: 12 / Enantiomer: LEVO / EC number: aspartyl aminopeptidase
Source (natural)Organism: Escherichia coli (strain K12) (bacteria)
Molecular weightTheoretical: 102.615375 KDa
Recombinant expressionOrganism: Schizosaccharomyces pombe 972h- (yeast)
SequenceString: MQLHGKMTAT AKSCALDFLD FVNASPTPYH AVQNLAEHYM SHGFQYLSEK SDWQSKIEPG NSYFVTRNKS SIIAFSIGKK WKPGNGFSI IATHTDSPTL RLKPKSQKSA YGYLQVGVEK YGGGIWHTWF DRDLSLAGRV MVEEEDGRVI QYNVHIDRPL L RIPTLAIH ...String:
MQLHGKMTAT AKSCALDFLD FVNASPTPYH AVQNLAEHYM SHGFQYLSEK SDWQSKIEPG NSYFVTRNKS SIIAFSIGKK WKPGNGFSI IATHTDSPTL RLKPKSQKSA YGYLQVGVEK YGGGIWHTWF DRDLSLAGRV MVEEEDGRVI QYNVHIDRPL L RIPTLAIH LDPSANSSFS FNMETEFVPL IGLENELAKE ETSDNGDKYH HPVLLSLLAN EISKSLETTI DPSKIVDFEL IL GDAEKAR LGGIHEEFVF SPRLDNLGMT FCASQALTKS LENNSLDNES CVRVVPSFDH EEIGSVSAQG AESTFLPAVL QRI CELGKE SSLFSISMVK SFLVSADMAH AMHPNYSSRY ENSNTPFLNK GTVIKVNANQ RYTTNSAGIV LLKKVAQLAD VPIQ SFVVR NDSPCGSTIG PKLAAMTGMR TLDLGNPMLS MHSCREMCGS KDFEYAVVLF SSFFQNFANL EEKIIIDEGF KKASS SDNK EQPTLRSSSV ACNTCLKIIR NDSFHCTKCF DFDVCRDCYA KQAFLHPCPK PHFVLVRSSI PSVASLTCSV NSMSVS PQS KIEEGKLVIW INGDKGYNGL AEVGKKFEKD TGIKVTVEHP DKLEEKFPQV AATGDGPDII FWAHDRFGGY AQSGLLA EI TPDKAFQDKL YPFTWDAVRY NGKLIAYPIA VEALSLIYNK DLLPNPPKTW EEIPALDKEL KAKGKSALMF NLQEPYFT W PLIAADGGYA FKYENGKYDI KDVGVDNAGA KAGLTFLVDL IKNKHMNADT DYSIAEAAFN KGETAMTING PWAWSNIDT SKVNYGVTVL PTFKGQPSKP FVGVLSAGIN AASPNKELAK EFLENYLLTD EGLEAVNKDK PLGAVALKSY EEELAKDPRI AATMENAQK GEIMPNIPQM SAFWYAVRTA VINAASGRQT VDEALKDAQT

UniProtKB: Aspartyl aminopeptidase 1, Autophagy receptor Nbr1, Maltose/maltodextrin-binding periplasmic protein

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 48 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 1911 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMTris-HClTris hydrochloride
150.0 mMNaClSodium chloride
5.0 mMMgCl2Magnesium chloride
GridModel: Homemade / Material: GOLD / Mesh: 400 / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Tridiem 4K / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 1799 / Average exposure time: 5.4 sec. / Average electron dose: 50.0 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: 1.8 µm / Nominal defocus min: 1.2 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

Startup modelType of model: EMDB MAP
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.26 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0-beta) / Number images used: 414202
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0-beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0-beta)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Correlation coefficient
Output model

PDB-7dde:
Cryo-EM structure of the Ape4 and Nbr1 complex

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