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- EMDB-20236: Structure of S. cerevisiae protein O-mannosyltransferase Pmt1-Pmt... -

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Basic information

Entry
Database: EMDB / ID: EMD-20236
TitleStructure of S. cerevisiae protein O-mannosyltransferase Pmt1-Pmt2 complex bound to the sugar donor and a peptide acceptor
Map data
SampleProtein O-mannosyl transferase complex
  • (Dolichyl-phosphate-mannose--protein mannosyltransferase ...) x 2
  • acceptor peptide
  • (ligand) x 3
Function / homology
Function and homology information


dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt3p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt5p-Pmt2p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase activity / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt2p dimer complex / protein exit from endoplasmic reticulum / protein O-linked mannosylation / regulation of endoplasmic reticulum unfolded protein response / ER-associated misfolded protein catabolic process / fungal-type cell wall organization ...dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt3p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt5p-Pmt2p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase activity / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt2p dimer complex / protein exit from endoplasmic reticulum / protein O-linked mannosylation / regulation of endoplasmic reticulum unfolded protein response / ER-associated misfolded protein catabolic process / fungal-type cell wall organization / protein O-linked glycosylation / endoplasmic reticulum membrane / endoplasmic reticulum / integral component of membrane
MIR domain profile. / C-terminal four TMM region of protein-O-mannosyltransferase / MIR domain / Dolichyl-phosphate-mannose-protein mannosyltransferase / Mir domain superfamily / Protein O-mannosyl-transferase, C-terminal four TM domain / Glycosyltransferase 39-like / MIR motif / Glycosyl transferase family 39/83
Dolichyl-phosphate-mannose--protein mannosyltransferase 2 / Dolichyl-phosphate-mannose--protein mannosyltransferase 1
Biological speciesSaccharomyces cerevisiae W303 (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBai L / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer InstituteR01-CA231466 United States
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2019
Title: Structure of the eukaryotic protein O-mannosyltransferase Pmt1-Pmt2 complex.
Authors: Lin Bai / Amanda Kovach / Qinglong You / Alanna Kenny / Huilin Li /
Abstract: In eukaryotes, a nascent peptide entering the endoplasmic reticulum (ER) is scanned by two Sec61 translocon-associated large membrane machines for protein N-glycosylation and protein O-mannosylation, ...In eukaryotes, a nascent peptide entering the endoplasmic reticulum (ER) is scanned by two Sec61 translocon-associated large membrane machines for protein N-glycosylation and protein O-mannosylation, respectively. While the structure of the eight-protein oligosaccharyltransferase complex has been determined recently, the structures of mannosyltransferases of the PMT family, which are an integral part of ER protein homeostasis, are still unknown. Here we report cryo-EM structures of the Saccharomyces cerevisiae Pmt1-Pmt2 complex bound to a donor and an acceptor peptide at 3.2-Å resolution, showing that each subunit contains 11 transmembrane helices and a lumenal β-trefoil fold termed the MIR domain. The structures reveal the substrate recognition model and confirm an inverting mannosyl-transferring reaction mechanism by the enzyme complex. Furthermore, we found that the transmembrane domains of Pmt1 and Pmt2 share a structural fold with the catalytic subunits of oligosaccharyltransferases, confirming a previously proposed evolutionary relationship between protein O-mannosylation and protein N-glycosylation.
Validation ReportPDB-ID: 6p25

SummaryFull reportAbout validation report
History
DepositionMay 21, 2019-
Header (metadata) releaseJun 5, 2019-
Map releaseJul 10, 2019-
UpdateAug 21, 2019-
Current statusAug 21, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6p25
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20236.map.gz / Format: CCP4 / Size: 37.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 214 pix.
= 220.206 Å
1.03 Å/pix.
x 214 pix.
= 220.206 Å
1.03 Å/pix.
x 214 pix.
= 220.206 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.029 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.19949104 - 0.259651
Average (Standard dev.)0.00052829494 (±0.0069904085)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions214214214
Spacing214214214
CellA=B=C: 220.20601 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0291.0291.029
M x/y/z214214214
origin x/y/z0.0000.0000.000
length x/y/z220.206220.206220.206
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS214214214
D min/max/mean-0.1990.2600.001

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Supplemental data

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Sample components

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Entire Protein O-mannosyl transferase complex

EntireName: Protein O-mannosyl transferase complex / Number of components: 7

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Component #1: protein, Protein O-mannosyl transferase complex

ProteinName: Protein O-mannosyl transferase complex / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae W303 (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #2: protein, Dolichyl-phosphate-mannose--protein mannosyltransferase 1

ProteinName: Dolichyl-phosphate-mannose--protein mannosyltransferase 1
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 92.771125 kDa
SourceSpecies: Saccharomyces cerevisiae W303 (yeast) / Strain: W303
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #3: protein, Dolichyl-phosphate-mannose--protein mannosyltransferase 2

ProteinName: Dolichyl-phosphate-mannose--protein mannosyltransferase 2
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 86.957422 kDa
SourceSpecies: Saccharomyces cerevisiae W303 (yeast) / Strain: W303
Source (engineered)Expression System: Saccharomyces cerevisiae W303 (yeast)

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Component #4: protein, acceptor peptide

ProteinName: acceptor peptide / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.478538 kDa
SourceSpecies: Saccharomyces cerevisiae W303 (yeast)

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Component #5: ligand, N-ACETYL-D-GLUCOSAMINE

LigandName: N-ACETYL-D-GLUCOSAMINEN-Acetylglucosamine / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.221208 kDa

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Component #6: ligand, 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

LigandName: 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.75806 kDa

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Component #7: ligand, (3R)-3,31-dimethyl-7,11,15,19,23,27-hexamethylidenedotria...

LigandName: (3R)-3,31-dimethyl-7,11,15,19,23,27-hexamethylidenedotriacont-31-en-1-yl dihydrogen phosphate
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.644947 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.4
Support filmunspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 2 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 521848
3D reconstructionSoftware: Coot, UCSF Chimera / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible
Output model

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