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- PDB-6p25: Structure of S. cerevisiae protein O-mannosyltransferase Pmt1-Pmt... -

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Basic information

Entry
Database: PDB / ID: 6p25
TitleStructure of S. cerevisiae protein O-mannosyltransferase Pmt1-Pmt2 complex bound to the sugar donor and a peptide acceptor
Components
  • (Dolichyl-phosphate-mannose--protein mannosyltransferase ...) x 2
  • acceptor peptide
KeywordsTRANSFERASE / complex / glycosylation
Function / homology
Function and homology information


dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt3p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt5p-Pmt2p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase / dolichyl-phosphate-mannose-protein mannosyltransferase activity / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt2p dimer complex / 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 Pmt5p-Pmt2p dimer complex / dolichyl-phosphate-mannose-protein mannosyltransferase / dolichyl-phosphate-mannose-protein mannosyltransferase activity / dolichyl-phosphate-mannose-protein mannosyltransferase Pmt1p-Pmt2p dimer complex / 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 / : / endoplasmic reticulum membrane / endoplasmic reticulum
Similarity search - Function
Dolichyl-phosphate-mannose-protein mannosyltransferase 1/5 / 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
Chem-CPL / Chem-NNM / Dolichyl-phosphate-mannose--protein mannosyltransferase 2 / Dolichyl-phosphate-mannose--protein mannosyltransferase 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae W303 (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBai, L. / Li, H.
Funding support United States, 1items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 10, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Aug 21, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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  • Deposited structure unit
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  • EMDB-20236
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Assembly

Deposited unit
A: Dolichyl-phosphate-mannose--protein mannosyltransferase 1
B: Dolichyl-phosphate-mannose--protein mannosyltransferase 2
D: acceptor peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)183,5699
Polymers180,2073
Non-polymers3,3626
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6950 Å2
ΔGint-34 kcal/mol
Surface area61240 Å2

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Components

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Dolichyl-phosphate-mannose--protein mannosyltransferase ... , 2 types, 2 molecules AB

#1: Protein Dolichyl-phosphate-mannose--protein mannosyltransferase 1 / Pmt1


Mass: 92771.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Strain: W303 / Gene: PMT1, YDL095W, D2390 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): W303
References: UniProt: P33775, dolichyl-phosphate-mannose-protein mannosyltransferase
#2: Protein Dolichyl-phosphate-mannose--protein mannosyltransferase 2 / Pmt2


Mass: 86957.422 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Strain: W303 / Gene: PMT2, FUN25, YAL023C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): W303
References: UniProt: P31382, dolichyl-phosphate-mannose-protein mannosyltransferase

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Protein/peptide / Sugars , 2 types, 3 molecules D

#3: Protein/peptide acceptor peptide


Mass: 478.538 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae W303 (yeast)
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-CPL / 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / PALMITOYL-LINOLEOYL PHOSPHATIDYLCHOLINE / Phosphatidylcholine


Mass: 758.060 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C42H80NO8P / Comment: phospholipid*YM
#6: Chemical ChemComp-NNM / (3R)-3,31-dimethyl-7,11,15,19,23,27-hexamethylidenedotriacont-31-en-1-yl dihydrogen phosphate


Mass: 644.947 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H69O4P / Feature type: SUBJECT OF INVESTIGATION

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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: Protein O-mannosyl transferase complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae W303 (yeast)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 2 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
12RELION2.1final Euler assignment
13RELIONclassification
14Coot3D reconstruction
15UCSF Chimera3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 953292
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 521848 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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