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- PDB-7dd9: Cryo-EM structure of the Ams1 and Nbr1 complex -

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Basic information

Entry
Database: PDB / ID: 7dd9
TitleCryo-EM structure of the Ams1 and Nbr1 complex
ComponentsAlpha-mannosidase,ZZ-type zinc finger-containing protein P35G2.11c,Maltose/maltodextrin-binding periplasmic protein
KeywordsHYDROLASE / glycoside hydrolase / signaling protein / autophagy
Function / homology
Function and homology information


mannosyl-oligosaccharide 1,6-alpha-mannosidase activity / mannosyl-oligosaccharide 1,3-alpha-mannosidase activity / NVT complex / Lysosomal oligosaccharide catabolism / Pexophagy / alpha-mannosidase / fungal-type vacuole lumen / cytoplasm to vacuole targeting by the NVT pathway / alpha-mannosidase activity / oligosaccharide catabolic process ...mannosyl-oligosaccharide 1,6-alpha-mannosidase activity / mannosyl-oligosaccharide 1,3-alpha-mannosidase activity / NVT complex / Lysosomal oligosaccharide catabolism / Pexophagy / alpha-mannosidase / fungal-type vacuole lumen / cytoplasm to vacuole targeting by the NVT pathway / alpha-mannosidase activity / oligosaccharide catabolic process / mannose metabolic process / fungal-type vacuole membrane / cargo receptor activity / detection of maltose stimulus / maltose binding / maltose transport complex / maltose transport / maltodextrin transmembrane transport / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / carbohydrate transport / ATP-binding cassette (ABC) transporter complex / multivesicular body / cell chemotaxis / outer membrane-bounded periplasmic space / carbohydrate binding / periplasmic space / DNA damage response / Golgi apparatus / zinc ion binding / membrane / metal ion binding / cytosol
Similarity search - Function
Next to BRCA1, central domain / Ig-like domain from next to BRCA1 gene / Glycosyl hydrolases family 38, C-terminal beta sandwich domain / Glycosyl hydrolases family 38 C-terminal beta sandwich domain / Glycoside hydrolase family 38, N-terminal domain / Glycosyl hydrolase family 38, C-terminal / Glycoside hydrolase family 38, central domain / Glycoside hydrolase family 38, central domain superfamily / Glycosyl hydrolases family 38 N-terminal domain / Glycosyl hydrolases family 38 C-terminal domain ...Next to BRCA1, central domain / Ig-like domain from next to BRCA1 gene / Glycosyl hydrolases family 38, C-terminal beta sandwich domain / Glycosyl hydrolases family 38 C-terminal beta sandwich domain / Glycoside hydrolase family 38, N-terminal domain / Glycosyl hydrolase family 38, C-terminal / Glycoside hydrolase family 38, central domain / Glycoside hydrolase family 38, central domain superfamily / Glycosyl hydrolases family 38 N-terminal domain / Glycosyl hydrolases family 38 C-terminal domain / Alpha mannosidase middle domain / Alpha mannosidase, middle domain / Glycoside hydrolase 38, N-terminal domain superfamily / Glycoside hydrolase families 57/38, central domain superfamily / Glycoside hydrolase/deacetylase, beta/alpha-barrel / 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. / Galactose mutarotase-like domain superfamily / 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
Maltose/maltodextrin-binding periplasmic protein / ZZ-type zinc finger-containing protein P35G2.11c / Alpha-mannosidase
Similarity search - Component
Biological speciesSchizosaccharomyces pombe (fission yeast)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsZhang, J. / Ye, K.
Funding support China, 3items
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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 14, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 11, 2021Group: Database references / Category: citation / database_2
Item: _citation.journal_volume / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Alpha-mannosidase,ZZ-type zinc finger-containing protein P35G2.11c,Maltose/maltodextrin-binding periplasmic protein
C: Alpha-mannosidase,ZZ-type zinc finger-containing protein P35G2.11c,Maltose/maltodextrin-binding periplasmic protein
E: Alpha-mannosidase,ZZ-type zinc finger-containing protein P35G2.11c,Maltose/maltodextrin-binding periplasmic protein
G: Alpha-mannosidase,ZZ-type zinc finger-containing protein P35G2.11c,Maltose/maltodextrin-binding periplasmic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)715,19016
Polymers714,4054
Non-polymers78512
Water17,096949
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Alpha-mannosidase,ZZ-type zinc finger-containing protein P35G2.11c,Maltose/maltodextrin-binding periplasmic protein / Alpha-D-mannoside mannohydrolase / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP


Mass: 178601.281 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: The fusion protein comprises of the full-length Ams1, a linker sequence (GFKKASSSDNKEQ), residues 53-180 of Nbr1, and maltose binding protein (MBP).
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast), (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: ams1, mns2, SPAC513.05, SPBP35G2.11c, malE, b4034, JW3994
Production host: Schizosaccharomyces pombe 972h- (yeast)
References: UniProt: Q9UT61, UniProt: Q9P792, UniProt: P0AEX9, alpha-mannosidase
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 949 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ams1 and Nbr1 fusion protein / Type: COMPLEX
Details: The fusion protein comprises of the full-length Ams1, a linker sequence (GFKKASSSDNKEQ), residues 53-180 of Nbr1, and maltose binding protein (MBP).
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.52 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Schizosaccharomyces pombe 972h- (yeast)284812
21Escherichia coli (strain K12) (bacteria)83333
Source (recombinant)Organism: Schizosaccharomyces pombe 972h- (yeast)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris hydrochlorideTris-HClTris1
2150 mMSodium chlorideNaClSodium chloride1
35 mMMagnesium chlorideMgCl21
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 5 seconds before plunging

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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 defocus max: 2200 nm / Nominal defocus min: 1600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8.9 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2430
EM imaging opticsEnergyfilter name: GIF Tridiem 4K / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 36

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
1Gautomatch0.53particle selection
2SerialEMimage acquisition
4Gctf1.06CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3.0-betainitial Euler assignment
11RELION3.0-betafinal Euler assignment
12RELION3.0-betaclassification
13RELION3.0-beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 296884 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 36.84 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007337124
ELECTRON MICROSCOPYf_angle_d0.701250372
ELECTRON MICROSCOPYf_chiral_restr0.05135464
ELECTRON MICROSCOPYf_plane_restr0.00396516
ELECTRON MICROSCOPYf_dihedral_angle_d12.12174836

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