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- EMDB-20240: Structure of S. cerevisiae protein O-mannosyltransferase Pmt1-Pmt... -

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Basic information

Entry
Database: EMDB / ID: EMD-20240
TitleStructure of S. cerevisiae protein O-mannosyltransferase Pmt1-Pmt2 complex bound to the sugar donor
Map dataO-mannosyltransferase Pmt1-Pmt2 complex bound to the sugar donor
Sample
  • Complex: Protein O-mannosyl transferase complex
    • Protein or peptide: Dolichyl-phosphate-mannose--protein mannosyltransferase 1
    • Protein or peptide: Dolichyl-phosphate-mannose--protein mannosyltransferase 2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: (3R)-3,31-dimethyl-7,11,15,19,23,27-hexamethylidenedotriacont-31-en-1-yl dihydrogen phosphate
Keywordscomplex / TRANSFERASE / glycosylation
Function / homology
Function and homology information


dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt3p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt2p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt5p-Pmt2p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase / dolichyl-phosphate-mannose-protein mannosyltransferase activity / regulation of endoplasmic reticulum unfolded protein response / fungal-type cell wall biogenesis / protein O-linked mannosylation / protein exit from endoplasmic reticulum / protein O-linked glycosylation ...dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt3p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt2p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt5p-Pmt2p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase / dolichyl-phosphate-mannose-protein mannosyltransferase activity / regulation of endoplasmic reticulum unfolded protein response / fungal-type cell wall biogenesis / protein O-linked mannosylation / protein exit from endoplasmic reticulum / protein O-linked glycosylation / ERAD pathway / endoplasmic reticulum membrane / endoplasmic reticulum
Similarity search - Function
Glycosyl transferase family 39/83 / Glycosyltransferase 39-like / Protein O-mannosyl-transferase, C-terminal four TM domain / Dolichyl-phosphate-mannose-protein mannosyltransferase / C-terminal four TMM region of protein-O-mannosyltransferase / MIR motif / MIR domain / MIR domain profile. / Domain in ryanodine and inositol trisphosphate receptors and protein O-mannosyltransferases / Mir domain superfamily
Similarity search - Domain/homology
Dolichyl-phosphate-mannose--protein mannosyltransferase 2 / Dolichyl-phosphate-mannose--protein mannosyltransferase 1
Similarity search - Component
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 Institute (NIH/NCI)R01-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.
History
DepositionMay 21, 2019-
Header (metadata) releaseJun 5, 2019-
Map releaseJul 10, 2019-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6p2r
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20240.png

Downloads & links

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Map

FileDownload / File: emd_20240.map.gz / Format: CCP4 / Size: 37.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationO-mannosyltransferase Pmt1-Pmt2 complex bound to the sugar donor
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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.12542087 - 0.18337071
Average (Standard dev.)0.00052411866 (±0.005234213)
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-0.1250.1830.001

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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
Components
  • Complex: Protein O-mannosyl transferase complex
    • Protein or peptide: Dolichyl-phosphate-mannose--protein mannosyltransferase 1
    • Protein or peptide: Dolichyl-phosphate-mannose--protein mannosyltransferase 2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: (3R)-3,31-dimethyl-7,11,15,19,23,27-hexamethylidenedotriacont-31-en-1-yl dihydrogen phosphate

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Supramolecule #1: Protein O-mannosyl transferase complex

SupramoleculeName: Protein O-mannosyl transferase complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)

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Macromolecule #1: Dolichyl-phosphate-mannose--protein mannosyltransferase 1

MacromoleculeName: Dolichyl-phosphate-mannose--protein mannosyltransferase 1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: dolichyl-phosphate-mannose-protein mannosyltransferase
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Molecular weightTheoretical: 92.771125 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MSEEKTYKRV EQDDPVPELD IKQGPVRPFI VTDPSAELAS LRTMVTLKEK LLVACLAVFT AVIRLHGLAW PDSVVFDEVH FGGFASQYI RGTYFMDVHP PLAKMLYAGV ASLGGFQGDF DFENIGDSFP STTPYVLMRF FSASLGALTV ILMYMTLRYS G VRMWVALM ...String:
MSEEKTYKRV EQDDPVPELD IKQGPVRPFI VTDPSAELAS LRTMVTLKEK LLVACLAVFT AVIRLHGLAW PDSVVFDEVH FGGFASQYI RGTYFMDVHP PLAKMLYAGV ASLGGFQGDF DFENIGDSFP STTPYVLMRF FSASLGALTV ILMYMTLRYS G VRMWVALM SAICFAVENS YVTISRYILL DAPLMFFIAA AVYSFKKYEM YPANSLNAYK SLLATGIALG MASSSKWVGL FT VTWVGLL CIWRLWFMIG DLTKSSKSIF KVAFAKLAFL LGVPFALYLV FFYIHFQSLT LDGDGASFFS PEFRSTLKNN KIP QNVVAD VGIGSIISLR HLSTMGGYLH SHSHNYPAGS EQQQSTLYPH MDANNDWLLE LYNAPGESLT TFQNLTDGTK VRLF HTVTR CRLHSHDHKP PVSESSDWQK EVSCYGYSGF DGDANDDWVV EIDKKNSAPG VAQERVIALD TKFRLRHAMT GCYLF SHEV KLPAWGFEQQ EVTCASSGRH DLTLWYVENN SNPLLPEDTK RISYKPASFI SKFIESHKKM WHINKNLVEP HVYESQ PTS WPFLLRGISY WGENNRNVYL LGNAIVWWAV TAFIGIFGLI VITELFSWQL GKPILKDSKV VNFHVQVIHY LLGFAVH YA PSFLMQRQMF LHHYLPAYYF GILALGHALD IIVSYVFRSK RQMGYAVVIT FLAASVYFFK SFSPIIYGTP WTQELCQK S QWLSGWDYNC NTYFSSLEEY KNQTLTKRES QPAATSTVEE ITIEGDGPSY EDLMNEDGKK IFKDTEGNEL DPEVVKKML EEEGANILKV EKRAVLE

UniProtKB: Dolichyl-phosphate-mannose--protein mannosyltransferase 1

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Macromolecule #2: Dolichyl-phosphate-mannose--protein mannosyltransferase 2

MacromoleculeName: Dolichyl-phosphate-mannose--protein mannosyltransferase 2
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: dolichyl-phosphate-mannose-protein mannosyltransferase
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Molecular weightTheoretical: 86.957422 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MSSSSSTGYS KNNAAHIKQE NTLRQRESSS ISVSEELSSA DERDAEDFSK EKPAAQSSLL RLESVVMPVI FTALALFTRM YKIGINNHV VWDEAHFGKF GSYYLRHEFY HDVHPPLGKM LVGLSGYLAG YNGSWDFPSG EIYPDYLDYV KMRLFNASFS A LCVPLAYF ...String:
MSSSSSTGYS KNNAAHIKQE NTLRQRESSS ISVSEELSSA DERDAEDFSK EKPAAQSSLL RLESVVMPVI FTALALFTRM YKIGINNHV VWDEAHFGKF GSYYLRHEFY HDVHPPLGKM LVGLSGYLAG YNGSWDFPSG EIYPDYLDYV KMRLFNASFS A LCVPLAYF TAKAIGFSLP TVWLMTVLVL FENSYSTLGR FILLDSMLLF FTVASFFSFV MFHNQRSKPF SRKWWKWLLI TG ISLGCTI SVKMVGLFII TMVGIYTVID LWTFLADKSM SWKTYINHWL ARIFGLIIVP FCIFLLCFKI HFDLLSHSGT GDA NMPSLF QARLVGSDVG QGPRDIALGS SVVSIKNQAL GGSLLHSHIQ TYPDGSNQQQ VTCYGYKDAN NEWFFNRERG LPSW SENET DIEYLKPGTS YRLVHKSTGR NLHTHPVAAP VSKTQWEVSG YGDNVVGDNK DNWVIEIMDQ RGDEDPEKLH TLTTS FRIK NLEMGCYLAQ TGNSLPEWGF RQQEVVCMKN PFKRDKRTWW NIETHENERL PPRPEDFQYP KTNFLKDFIH LNLAMM ATN NALVPDPDKF DYLASSAWQW PTLNVGLRLC GWGDDNPKYF LLGTPASTWA SSVAVLAFMA TVVILLIRWQ RQYVDLR NP SNWNVFLMGG FYPLLAWGLH YMPFVIMSRV TYVHHYLPAL YFALIILAYC FDAGLQKWSR SKCGRIMRFV LYAGFMAL V IGCFWYFSPI SFGMEGPSSN FRYLNWFSTW DIADKQEA

UniProtKB: Dolichyl-phosphate-mannose--protein mannosyltransferase 2

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #4: 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 4 / Number of copies: 3 / Formula: CPL
Molecular weightTheoretical: 758.06 Da
Chemical component information

ChemComp-CPL:
1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM

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Macromolecule #5: (3R)-3,31-dimethyl-7,11,15,19,23,27-hexamethylidenedotriacont-31-...

MacromoleculeName: (3R)-3,31-dimethyl-7,11,15,19,23,27-hexamethylidenedotriacont-31-en-1-yl dihydrogen phosphate
type: ligand / ID: 5 / Number of copies: 1 / Formula: NNM
Molecular weightTheoretical: 644.947 Da
Chemical component information

ChemComp-NNM:
(3R)-3,31-dimethyl-7,11,15,19,23,27-hexamethylidenedotriacont-31-en-1-yl dihydrogen phosphate

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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

BufferpH: 7.4
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 913730
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: Coot, UCSF Chimera) / Number images used: 581966
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final 3D classificationSoftware - Name: RELION

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6p2r:
Structure of S. cerevisiae protein O-mannosyltransferase Pmt1-Pmt2 complex bound to the sugar donor

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