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- EMDB-8166: Structure of the S. cerevisiae alpha-mannosidase 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-8166
TitleStructure of the S. cerevisiae alpha-mannosidase 1
Map dataNone
Sample
  • Complex: Alpha-mannosidase 1
    • Protein or peptide: Alpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase
Function / homology
Function and homology information


mannose catabolic process / Lysosomal oligosaccharide catabolism / alpha-mannosidase / Cvt complex / alpha-mannosidase activity / oligosaccharide catabolic process / fungal-type vacuole membrane / carbohydrate binding / metal ion binding
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 speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae S288c (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsSchneider S / Kosinski J / Jakobi AJ / Hagen WJH / 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, 2016-
Header (metadata) releaseMay 25, 2016-
Map releaseJun 15, 2016-
UpdateAug 2, 2017-
Current statusAug 2, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.064
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.064
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5jm0
  • Surface level: 0.064
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5jm0
  • Surface level: 0.064
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8166.png

Downloads & links

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Map

FileDownload / File: emd_8166.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 250 pix.
= 270. Å		1.08 Å/pix.
x 250 pix.
= 270. Å		1.08 Å/pix.
x 250 pix.
= 270. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.064 / Movie #1: 0.064
Minimum - Maximum-0.032903958 - 0.13467872
Average (Standard dev.)0.0019863124 (±0.010677306)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.0330.1350.002

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Supplemental data

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Sample components

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Entire : Alpha-mannosidase 1

EntireName: Alpha-mannosidase 1
Components
  • Complex: Alpha-mannosidase 1
    • Protein or peptide: Alpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase

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Supramolecule #1: Alpha-mannosidase 1

SupramoleculeName: Alpha-mannosidase 1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: His-tagged
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: S288c
Recombinant expressionOrganism: Komagataella pastoris (fungus) / Recombinant strain: SMD1168 / Recombinant plasmid: pGAPz
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: Alpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase

MacromoleculeName: Alpha-mannosidase,Alpha-mannosidase,Alpha-mannosidase / type: protein_or_peptide / ID: 1
Details: The coordinate model contains a poly-alanine stretch that corresponds to residues 17-27 in the template sequence.
Number of copies: 1 / Enantiomer: LEVO / EC number: alpha-mannosidase
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast)
Molecular weightTheoretical: 125.764156 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)MSSEDII YDPQFKPV Q GIYENRLRQF IDTGGDYHDL NLPKFYDKKR ISLDHDHVKV WWYQVSFERG SSPVSPDKRP SWKSIIERDK KGELEFREA NINQPFGPSW ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)MSSEDII YDPQFKPV Q GIYENRLRQF IDTGGDYHDL NLPKFYDKKR ISLDHDHVKV WWYQVSFERG SSPVSPDKRP SWKSIIERDK KGELEFREA NINQPFGPSW STTWFKVKIS LPEDWVKSNE QLLFQWDCSN EGIVIDPKTL IPVTAFSGGE RTEYVLPKTS DGKHFFYIEA GNNGMFGCG AGSTINPPDD NRFFHLRKAD IVWPDLDARA LYIDFWMLGD AARELPGDSW QKHQARQLGN AVMNLFDPND R SSVRKCRE LLQREYFDSF LESSKVYEQG ESQVLTNVYG IGNCHIDTAW LWPFAETRRK IVRSWSSQCT LMDRFPEYKF VA SQAQQFK WLLEDHPEFF NKVLIPKIQQ SQFFAVGGTW VENDTNIPSG ESLARQFFFG QRFFLKHFGL KSKIFWLPDT FGY SSQMPQ LCRLSGIDKF LTQKLSWNNI NSFPHSTFNW AGIDGSQLLT HMPPGNTYTA DSHFGDVLRT AKQNKTPEYY GSGL MLYGK GDGGGGPTEE MLQKMRRIRS MNNRNGNVIP KLQVGITVDE FYDDILKRTN QGHDLPTWSG ELYFEFHRGT YTSQA QTKK LMRLSEIKLH DLEWIAAKTS VLYPDSYKYP SKQINELWEN VLLCQFHDVL PGSCIEMVYK YEAVPMLHNV VKECTS LID KTVQFLQSQS KADLVEMRTL TWSKPEKVSE ECSLNGSYTS SVTGYDDYIV LANGKLKVII CKKTGVITSI TDETLGV EY LDTEHGRNKL GANQFVIYDD KPLGWQAWDT ELYSVNQYKY VTKPKKVQVS CNTKEKCAVE VIFQISEKCK IKSVISLN A TAVTDAKLSK VDISTTVENW DARNKFLKVE FPVNIRNDFA SYETQFGITK RPTHYNTSWD VAKFEVCHHK FADYSEYSK GVSILNDCKY GFSTHGNLMR LSLLRSPKAP DAHADMGTHE IKYAIYPHRG ALSSDTVKLA HEFNYCFKYK LPKDIGMNFD DIISISGDE NVILSNIKRG EDDSAVKSNY SLNPRDEQSI VVRVYESLGG ESFASLNTTL NLKRIEKVDN LEMKVYKSLT A TRDESNHA INRIPIKLRP FEIASFRLYF

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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

Concentration0.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMTris-HClTris
175.0 mMNaClSodium chlorideSodium chloride
75.0 mMImidazoleImidazole
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 4.5 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK III
Details: 2.5 ul of sample was applied, offset -3, 9s blotting time.
Detailssingle particles alongside chains of tetramers

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Frames/image: 1-7 / Number grids imaged: 1 / Number real images: 1735 / Average exposure time: 1.6 sec. / Average electron dose: 58.0 e/Å2
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.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 85274
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 3)determine
RELION (ver. 1.3)apply
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Number images: 73 / Random conical tilt - Tilt angle: 55 degrees / Details: EMAN2
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN (ver. 2.06)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3)
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.
Number images used: 33588
FSC plot (resolution estimation)

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Atomic model buiding 1

Details1. 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
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 80 / Target criteria: Cross-correlation coefficient
Output model

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

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