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- PDB-5jm0: Structure of the S. cerevisiae alpha-mannosidase 1 -

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Basic information

Entry
Database: PDB / ID: 5jm0
TitleStructure of the S. cerevisiae alpha-mannosidase 1
ComponentsAlpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase
KeywordsHYDROLASE / tetramer / cvt cargo / mannosidase / selective autophagy
Function / homology
Function and homology information


mannose catabolic process / Lysosomal oligosaccharide catabolism / Cvt complex / alpha-mannosidase / alpha-mannosidase activity / fungal-type vacuole membrane / oligosaccharide catabolic process / cellular response to nitrogen starvation / cellular response to glucose starvation / carbohydrate binding / metal ion binding
Similarity search - Function
: / Alpha-mannosidase, jelly roll domain / 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, jelly roll domain / 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 families 57/38, central domain superfamily / Glycoside hydrolase 38, N-terminal domain superfamily / Glycoside hydrolase/deacetylase, beta/alpha-barrel / Galactose mutarotase-like domain superfamily
Similarity search - Domain/homology
Biological speciesSaccharomyces cerevisiae S288c (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsSchneider, S. / Kosinski, J. / Jakobi, A.J. / Hagen, W.J.H. / Sachse, C.
CitationJournal: EMBO Rep / Year: 2016
Title: Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle.
Authors: Chiara Bertipaglia / Sarah Schneider / Arjen J Jakobi / Abul K Tarafder / Yury S Bykov / Andrea Picco / Wanda Kukulski / Jan Kosinski / Wim Jh Hagen / Arvind C Ravichandran / Matthias ...Authors: Chiara Bertipaglia / Sarah Schneider / Arjen J Jakobi / Abul K Tarafder / Yury S Bykov / Andrea Picco / Wanda Kukulski / Jan Kosinski / Wim Jh Hagen / Arvind C Ravichandran / Matthias Wilmanns / Marko Kaksonen / John Ag Briggs / Carsten Sachse /
Abstract: Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles ...Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.
History
DepositionApr 28, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jun 15, 2016Provider: repository / Type: Initial release
Revision 1.1Jun 22, 2016Group: Database references
Revision 1.2Jun 29, 2016Group: Database references
Revision 1.3Jul 13, 2016Group: Database references
Revision 1.4Aug 2, 2017Group: Data collection / Experimental preparation / Refinement description
Category: em_3d_fitting / em_sample_support / em_software
Item: _em_3d_fitting.target_criteria / _em_sample_support.grid_type / _em_software.name
Revision 1.5May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Alpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase


Theoretical massNumber of molelcules
Total (without water)125,7641
Polymers125,7641
Non-polymers00
Water00
1
A: Alpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase

A: Alpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase

A: Alpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase

A: Alpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase


Theoretical massNumber of molelcules
Total (without water)503,0574
Polymers503,0574
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation3
MethodPISA
SymmetryPoint symmetry: (Schoenflies symbol: D2 (2x2 fold dihedral))

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Components

#1: Protein Alpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase / Alpha-D-mannoside mannohydrolase


Mass: 125764.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The coordinate model contains a poly-alanine stretch that corresponds to residues 17-27 in the template sequence.
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: AMS1, YGL156W, G1861 / Production host: Komagataella pastoris (fungus) / Strain (production host): SMD1168 / References: UniProt: P22855, alpha-mannosidase

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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: Alpha-mannosidase 1 / Type: COMPLEX / Details: His-tagged / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: S288c / Cellular location: cytoplasm, vacuole
Source (recombinant)Organism: Komagataella pastoris (fungus) / Strain: SMD1168 / Plasmid: pGAPz
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HCl1
2175 mMSodium chlorideNaCl1
375 mMImidazoleImidazole1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: single particles alongside chains of tetramers
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293 K
Details: 2.5 ul of sample was applied, offset -3, 9s blotting time

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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 FIELD / Nominal magnification: 75000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.6 sec. / Electron dose: 58 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1735
Details: The first six frames comprised each 7 e/A2 and the final frame consequently received 16 e/A2 dose. Relion's particle polishing procedure was used.
Image scansMovie frames/image: 7 / Used frames/image: 1-7

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Processing

EM software
IDNameVersionCategoryDetails
1RELION1.3particle selectionautopicking
2EPU1image acquisition
4CTFFIND3CTF correctiondetermine
5RELION1.3CTF correctionapply
8UCSF Chimera8.6model fitting
10EMAN2.06initial Euler assignment
11RELION1.3final Euler assignment
12RELION1.3classification
13RELION1.33D reconstruction
14DireXv0.5model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 85274
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33588 / Algorithm: FOURIER SPACE
Details: 1. From 85274 initially auto-picked particles, 16678 were eliminated after 2D classification. 2. From 68596, two classes making up 33588 particles were included in the final reconstruction.
Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 80 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: 1. Fitting of a homology model based on PDB 2wyh combined with a homology model based on PDB 2xbz and 3cmg for the C- (residues 287-1083) and N-terminal part (residues 45-203) respectively 2. ...Details: 1. Fitting of a homology model based on PDB 2wyh combined with a homology model based on PDB 2xbz and 3cmg for the C- (residues 287-1083) and N-terminal part (residues 45-203) respectively 2. ab initio modelling for a three-helix bundle of the N-terminal part (residues 209-286) 3. creating an ideal poly-alanine helix for residues 17-27

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