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- PDB-6c26: The Cryo-EM structure of a eukaryotic oligosaccharyl transferase ... -

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Entry
Database: PDB / ID: 6c26
TitleThe Cryo-EM structure of a eukaryotic oligosaccharyl transferase complex
Components(Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ...) x 8
KeywordsTRANSFERASE / complex / glycosylation
Function / homology
Function and homology information


Miscellaneous transport and binding events / oligosaccharyltransferase I complex / protein O-linked mannosylation / oligosaccharyltransferase complex / dolichyl-diphosphooligosaccharide-protein glycotransferase / dolichyl-diphosphooligosaccharide-protein glycotransferase activity / protein N-linked glycosylation via asparagine / protein N-linked glycosylation / glycosyltransferase activity / protein glycosylation ...Miscellaneous transport and binding events / oligosaccharyltransferase I complex / protein O-linked mannosylation / oligosaccharyltransferase complex / dolichyl-diphosphooligosaccharide-protein glycotransferase / dolichyl-diphosphooligosaccharide-protein glycotransferase activity / protein N-linked glycosylation via asparagine / protein N-linked glycosylation / glycosyltransferase activity / protein glycosylation / protein-disulfide reductase activity / Neutrophil degranulation / post-translational protein modification / protein-macromolecule adaptor activity / nuclear envelope / protein-containing complex assembly / endoplasmic reticulum membrane / endoplasmic reticulum / mitochondrion / membrane / metal ion binding
Similarity search - Function
DAD/Ost2 / Oligosaccharyltransferase complex subunit / DAD family / Oligosaccharyltransferase subunit 5 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48kDa subunit / Oligosaccharyltransferase 48 kDa subunit beta / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit Swp1 / Oligosaccharyltransferase subunit Ribophorin II / Oligosaccaryltransferase / Oligosaccharyltransferase subunit ost4p superfamily ...DAD/Ost2 / Oligosaccharyltransferase complex subunit / DAD family / Oligosaccharyltransferase subunit 5 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48kDa subunit / Oligosaccharyltransferase 48 kDa subunit beta / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit Swp1 / Oligosaccharyltransferase subunit Ribophorin II / Oligosaccaryltransferase / Oligosaccharyltransferase subunit ost4p superfamily / Oligosaccaryltransferase / Ribophorin I / Ribophorin I / Oligosaccharyl transferase complex, subunit OST3/OST6 / OST3 / OST6 family, transporter family / : / STT3/PglB/AglB core domain / : / Oligosaccharyl transferase, STT3 subunit / Oligosaccharyl transferase STT3, N-terminal
Similarity search - Domain/homology
Chem-EGY / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit WBP1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 3 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit SWP1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST5 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBai, L. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM111742 United States
CitationJournal: Nature / Year: 2018
Title: The atomic structure of a eukaryotic oligosaccharyltransferase complex.
Authors: Lin Bai / Tong Wang / Gongpu Zhao / Amanda Kovach / Huilin Li /
Abstract: N-glycosylation is a ubiquitous modification of eukaryotic secretory and membrane-bound proteins; about 90% of glycoproteins are N-glycosylated. The reaction is catalysed by an eight-protein ...N-glycosylation is a ubiquitous modification of eukaryotic secretory and membrane-bound proteins; about 90% of glycoproteins are N-glycosylated. The reaction is catalysed by an eight-protein oligosaccharyltransferase (OST) complex that is embedded in the endoplasmic reticulum membrane. Our understanding of eukaryotic protein N-glycosylation has been limited owing to the lack of high-resolution structures. Here we report a 3.5 Å resolution cryo-electron microscopy structure of the Saccharomyces cerevisiae OST complex, revealing the structures of subunits Ost1-Ost5, Stt3, Wbp1 and Swp1. We found that seven phospholipids mediate many of the inter-subunit interactions, and an Stt3 N-glycan mediates interactions with Wbp1 and Swp1 in the lumen. Ost3 was found to mediate the OST-Sec61 translocon interface, funnelling the acceptor peptide towards the OST catalytic site as the nascent peptide emerges from the translocon. The structure provides insights into co-translational protein N-glycosylation, and may facilitate the development of small-molecule inhibitors that target this process.
History
DepositionJan 6, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2018Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Mar 7, 2018Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Mar 28, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / cell / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _pdbx_audit_support.funding_organization
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_validate_close_contact / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.pdbx_formal_charge / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_validate_close_contact.auth_asym_id_2 / _pdbx_validate_close_contact.auth_seq_id_2 / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.pdbx_value_order / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3
1: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1
5: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST5
4: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST4
2: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST2
3: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 3
C: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit SWP1
B: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit WBP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)291,72919
Polymers284,5918
Non-polymers7,13811
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ... , 8 types, 8 molecules A15423CB

#1: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3 / Oligosaccharyl transferase subunit STT3


Mass: 81604.539 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
References: UniProt: P39007, dolichyl-diphosphooligosaccharide-protein glycotransferase
#2: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Oligosaccharyl transferase 64 kDa subunit / Oligosaccharyl transferase subunit OST1 / ...Oligosaccharyl transferase 64 kDa subunit / Oligosaccharyl transferase subunit OST1 / Oligosaccharyl transferase subunit alpha


Mass: 54116.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
References: UniProt: P41543, dolichyl-diphosphooligosaccharide-protein glycotransferase
#3: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST5 / Oligosaccharyl transferase subunit OST5 / Oligosaccharyl transferase 9.5 kDa subunit / ...Oligosaccharyl transferase subunit OST5 / Oligosaccharyl transferase 9.5 kDa subunit / Oligosaccharyl transferase subunit zeta


Mass: 9525.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
References: UniProt: Q92316, dolichyl-diphosphooligosaccharide-protein glycotransferase
#4: Protein/peptide Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST4 / Oligosaccharyl transferase subunit OST4 / Oligosaccharyl transferase 4 kDa subunit / OTase 4 kDa subunit


Mass: 3986.696 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
References: UniProt: Q99380, dolichyl-diphosphooligosaccharide-protein glycotransferase
#5: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST2 / Oligosaccharyl transferase subunit OST2 / Oligosaccharyl transferase 16 kDa subunit / ...Oligosaccharyl transferase subunit OST2 / Oligosaccharyl transferase 16 kDa subunit / Oligosaccharyl transferase subunit epsilon


Mass: 14712.531 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
References: UniProt: P46964, dolichyl-diphosphooligosaccharide-protein glycotransferase
#6: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 3 / Oligosaccharyl transferase 34 kDa subunit / Oligosaccharyl transferase subunit OST3 / ...Oligosaccharyl transferase 34 kDa subunit / Oligosaccharyl transferase subunit OST3 / Oligosaccharyl transferase subunit gamma


Mass: 39518.160 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
References: UniProt: P48439, dolichyl-diphosphooligosaccharide-protein glycotransferase
#7: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit SWP1 / Oligosaccharyl transferase subunit SWP1 / Oligosaccharyl transferase subunit delta


Mass: 31682.832 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
References: UniProt: Q02795, dolichyl-diphosphooligosaccharide-protein glycotransferase
#8: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit WBP1 / Oligosaccharyl transferase subunit WBP1 / Oligosaccharyl transferase subunit beta


Mass: 49444.438 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
References: UniProt: P33767, dolichyl-diphosphooligosaccharide-protein glycotransferase

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Sugars , 3 types, 3 molecules

#9: Polysaccharide beta-D-mannopyranose-(1-2)-beta-D-mannopyranose-(1-2)-beta-D-mannopyranose-(1-2)-[beta-D- ...beta-D-mannopyranose-(1-2)-beta-D-mannopyranose-(1-2)-beta-D-mannopyranose-(1-2)-[beta-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1235.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-2DManpb1-2DManpb1-2[DManpb1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,7,6/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2-2-2-2-2/a4-b1_b4-c1_c2-d1_c6-g1_d2-e1_e2-f1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(2+1)][b-D-Manp]{[(2+1)][b-D-Manp]{[(2+1)][b-D-Manp]{}}}[(6+1)][b-D-Manp]{}}}}}LINUCSPDB-CARE
#10: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#12: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1
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 , 1 types, 8 molecules

#11: Chemical
ChemComp-EGY / (4R,7R)-4-hydroxy-N,N,N-trimethyl-4,9-dioxo-7-[(undecanoyloxy)methyl]-3,5,8-trioxa-4lambda~5~-phosphadocosan-1-aminium


Mass: 636.861 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C33H67NO8P / Comment: phospholipid*YM

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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: oligosaccharyl transferase complexOligosaccharyltransferase
Type: COMPLEX / Entity ID: #1-#8 / Source: NATURAL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: S288c
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
10RELIONfinal Euler assignment
11RELIONclassification
12Coot3D reconstruction
13UCSF Chimera3D reconstruction
14PHENIXmodel refinement
15Cootmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 823255
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 282202 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0117584
ELECTRON MICROSCOPYf_angle_d1.24123844
ELECTRON MICROSCOPYf_dihedral_angle_d9.7710289
ELECTRON MICROSCOPYf_chiral_restr0.0682685
ELECTRON MICROSCOPYf_plane_restr0.0082919

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