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- EMDB-30021: Structure of S.pombe alpha-mannosidase Ams1 -

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Basic information

Entry
Database: EMDB / ID: EMD-30021
TitleStructure of S.pombe alpha-mannosidase Ams1
Map data
Sample
  • Complex: Ams1 tetramer
    • Protein or peptide: Ams1
  • Ligand: ZINC ION
Keywordsglycoside hydrolase / HYDROLASE
Function / homology
Function and homology information


mannosyl-oligosaccharide 1,6-alpha-mannosidase activity / mannosyl-oligosaccharide 1,3-alpha-mannosidase activity / Lysosomal oligosaccharide catabolism / alpha-mannosidase / fungal-type vacuole lumen / alpha-mannosidase activity / mannose metabolic process / oligosaccharide catabolic process / fungal-type vacuole membrane / carbohydrate binding ...mannosyl-oligosaccharide 1,6-alpha-mannosidase activity / mannosyl-oligosaccharide 1,3-alpha-mannosidase activity / Lysosomal oligosaccharide catabolism / alpha-mannosidase / fungal-type vacuole lumen / alpha-mannosidase activity / mannose metabolic process / oligosaccharide catabolic process / fungal-type vacuole membrane / carbohydrate binding / metal ion binding / cytosol
Similarity search - Function
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 ...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 / Galactose mutarotase-like domain superfamily
Similarity search - Domain/homology
Biological speciesSchizosaccharomyces pombe (fission yeast) / Schizosaccharomyces pombe 972h- (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZhang J / Ye K
Funding support China, 3 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2017YFA0504600 China
National Natural Science Foundation of China (NSFC)91940302, 91540201, 31430024, 31325007 China
Chinese Academy of SciencesXDB37010201 China
CitationJournal: FEBS Open Bio / Year: 2020
Title: Cryo-EM structure of fission yeast tetrameric α-mannosidase Ams1.
Authors: Jianxiu Zhang / Ying-Ying Wang / Li-Lin Du / Keqiong Ye /
Abstract: Fungal α-mannosidase Ams1 and its mammalian homolog MAN2C1 hydrolyze terminal α-linked mannoses in free oligosaccharides released from misfolded glycoproteins or lipid-linked oligosaccharide donors. ...Fungal α-mannosidase Ams1 and its mammalian homolog MAN2C1 hydrolyze terminal α-linked mannoses in free oligosaccharides released from misfolded glycoproteins or lipid-linked oligosaccharide donors. Ams1 is transported by selective autophagy into vacuoles. Here, we determine the tetrameric structure of Ams1 from the fission yeast Schizosaccharomyces pombe at 3.2 Å resolution by cryo-electron microscopy. Distinct from a low resolution structure of S. cerevisiae Ams1, S. pombe Ams1 has a prominent N-terminal tail that mediates tetramerization and an extra β-sheet domain. Ams1 shares a conserved active site with other enzymes in glycoside hydrolase family 38, to which Ams1 belongs, but contains extra N-terminal domains involved in tetramerization. The atomic structure of Ams1 reported here will aid understanding of its enzymatic activity and transport mechanism.
History
DepositionFeb 17, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
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  • Surface view colored by radius
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-6lz1
  • Surface level: 0.05
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30021.png

Downloads & links

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Map

FileDownload / File: emd_30021.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.05
Minimum - Maximum-0.12418187 - 0.24946123
Average (Standard dev.)0.0013453268 (±0.01086885)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 249.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z249.600249.600249.600
α/β/γ90.00090.00090.000
start NX/NY/NZ107107101
NX/NY/NZ267267267
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1240.2490.001

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Supplemental data

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Mask #1

Fileemd_30021_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_30021_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_30021_half_map_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 : Ams1 tetramer

EntireName: Ams1 tetramer
Components
  • Complex: Ams1 tetramer
    • Protein or peptide: Ams1
  • Ligand: ZINC ION

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Supramolecule #1: Ams1 tetramer

SupramoleculeName: Ams1 tetramer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
Molecular weightTheoretical: 480 KDa

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Macromolecule #1: Ams1

MacromoleculeName: Ams1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: alpha-mannosidase
Source (natural)Organism: Schizosaccharomyces pombe 972h- (yeast)
Molecular weightTheoretical: 129.201672 KDa
Recombinant expressionOrganism: Schizosaccharomyces pombe (fission yeast)
SequenceString: MTLFPVLNNT PVGKQVDSIY ESRLDQFLSE GQYRDFNLPS VYDHARIDNP SGDVNNDLSK GFVDLKVYRV PDLSRPSFNE VVGHKKFDE TASKGDTFGP SWATFWFEVH IRLPKSWAKY EQVIFQWNCD NEGLVYSQDG VPLQAFSGSE RTDFILPDSW K TTEDTFYI ...String:
MTLFPVLNNT PVGKQVDSIY ESRLDQFLSE GQYRDFNLPS VYDHARIDNP SGDVNNDLSK GFVDLKVYRV PDLSRPSFNE VVGHKKFDE TASKGDTFGP SWATFWFEVH IRLPKSWAKY EQVIFQWNCD NEGLVYSQDG VPLQAFSGSE RTDFILPDSW K TTEDTFYI EMACNGMFGT GAGSQIAPPD PNRYFTLTKA DLVAPNLPAM ALAYDFLLMQ QCVKQLPSNC WQKYKARQIC ND IMNTFHP NDLSTINECR NLAKAFLGND IDSEAVFEKN NDKANVFAIG HCHIDTAWLW PFAETRRKIV RSWATQMNIM DRY PEYQFV CSQALQYLWL KEDHPDVFEK LKEYVNQNKF IPIGGSWVEH DTNIPNGESL IRQFLLGQHF FEKEFGVRCR TFWL PDTFG YSSQIPQICR LCGMDRFLTQ KLSWNNINSF PTSTFNWVAL DGSQVICHMP PANTYTADTN VNDVLHSIDQ HKNLV NDQA GLLVFGIGDG GGGPTPEMLE KLRRCKGIAN TVGYLPNVKL GNTVDEFFDG ILKRTNAGQT LPSWNGELYF EFHRGT YTT QAELKKLMRK VEIALHDAEY VSTLASIFSK DYSYPKESLQ DLWRDTLLCQ FHDVLPGSCI EMVYKDAIPI MSKVLKN TE ALLWQAIEQL GFKKASSSDN KEQLCLLNTL PWNVRGVITE TEENKLVYFE SCDGKGILTA AHTSLKHPAA AYQKDDNF I LVNDHLRVTI APNGLILSLF DLHKEREILD LKSGKNHAGA NQYVLFEDTP LSWQAWDTEV FSLEKYEVLD KGKVSIKES GPLRASVVVD IPISELSHMK ATISLEGYND CSEFTGVNFT CEVDWHESCK FLKVEFPVDI HSEFASYETQ FGITKRPTHY NTSWDVAKF EVCHQKFADY SDFTYGVSVL NDCKYGFSTH GNLMRLSLLR SPKQPDAHAD MGKHTIRYAV YPHSKPLDSS T VRAAHKFN SNFRLLTRAS DTANLDIFDA FQLVGEPNVI LSHIKMAEKG KSIILRVYES LGGKSRARLV IKSLTVASVT KC NGLEEDL EELCTLKSND YYEVPIELRA FEIATFKVNL GSMSKGKVVD IMDYKDDDDK PMDYKDDDDK HHHHHHDELY KRS GAPLLN KRISYDL

UniProtKB: Alpha-mannosidase

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMTris-HClTris hydrochloride
150.0 mMNaClSodium chloride
5.0 mMMgCl2Magnesium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / 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 opticsPhase plate: VOLTA PHASE PLATE / Energy 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: 597 / Average exposure time: 8.4 sec. / Average electron dose: 60.0 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: 1.0 µm / Nominal defocus min: 0.4 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
EMDB ID:

8166


Details: S. cerevisiae Ams1
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 75460
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)

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Atomic model buiding 1

Initial modelPDB ID:

6lz1


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Correlation coefficient
Output model

PDB-6lz1:
Structure of S.pombe alpha-mannosidase Ams1

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