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- PDB-8age: Structure of yeast oligosaccharylransferase complex with acceptor... -

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Basic information

Entry
Database: PDB / ID: 8age
TitleStructure of yeast oligosaccharylransferase complex with acceptor peptide bound
Components
  • (Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ...) x 6
  • OST3 isoform 1
  • OST4 isoform 1
  • PEPTIDE
KeywordsTRANSFERASE / N-glycosylation OST complex
Function / homology
Function and homology information


oligosaccharyltransferase I complex / oligosaccharyltransferase complex / dolichyl-diphosphooligosaccharide-protein glycotransferase / dolichyl-diphosphooligosaccharide-protein glycotransferase activity / protein N-linked glycosylation via asparagine / protein N-linked glycosylation / post-translational protein modification / endoplasmic reticulum membrane / endoplasmic reticulum / 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-323 / Chem-CPL / Chem-KZB / : / PHOSPHATIDYLETHANOLAMINE / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / OST3 isoform 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit WBP1 ...Chem-323 / Chem-CPL / Chem-KZB / : / PHOSPHATIDYLETHANOLAMINE / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / OST3 isoform 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit WBP1 / OST4 isoform 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST5
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsRamirez, A.S. / de Capitani, M. / Pesciullesi, G. / Kowal, J. / Bloch, J.S. / Irobalieva, R.N. / Aebi, M. / Reymond, J.L. / Locher, K.P.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Nat Commun / Year: 2022
Title: Molecular basis for glycan recognition and reaction priming of eukaryotic oligosaccharyltransferase.
Authors: Ana S Ramírez / Mario de Capitani / Giorgio Pesciullesi / Julia Kowal / Joël S Bloch / Rossitza N Irobalieva / Jean-Louis Reymond / Markus Aebi / Kaspar P Locher /
Abstract: Oligosaccharyltransferase (OST) is the central enzyme of N-linked protein glycosylation. It catalyzes the transfer of a pre-assembled glycan, GlcNAcManGlc, from a dolichyl-pyrophosphate donor to ...Oligosaccharyltransferase (OST) is the central enzyme of N-linked protein glycosylation. It catalyzes the transfer of a pre-assembled glycan, GlcNAcManGlc, from a dolichyl-pyrophosphate donor to acceptor sites in secretory proteins in the lumen of the endoplasmic reticulum. Precise recognition of the fully assembled glycan by OST is essential for the subsequent quality control steps of glycoprotein biosynthesis. However, the molecular basis of the OST-donor glycan interaction is unknown. Here we present cryo-EM structures of S. cerevisiae OST in distinct functional states. Our findings reveal that the terminal glucoses (Glc) of a chemo-enzymatically generated donor glycan analog bind to a pocket formed by the non-catalytic subunits WBP1 and OST2. We further find that binding either donor or acceptor substrate leads to distinct primed states of OST, where subsequent binding of the other substrate triggers conformational changes required for catalysis. This alternate priming allows OST to efficiently process closely spaced N-glycosylation sites.
History
DepositionJul 19, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 7, 2022Provider: repository / Type: Initial release
Revision 2.0Oct 18, 2023Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Non-polymer description / Source and taxonomy / Structure summary
Category: atom_site / atom_type ...atom_site / atom_type / chem_comp / chem_comp_atom / chem_comp_bond / em_entity_assembly / em_software / entity / entity_name_com / entity_src_gen / pdbx_branch_scheme / pdbx_contact_author / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_instance_feature / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop / pdbx_struct_conn_angle / pdbx_unobs_or_zero_occ_atoms / struct_asym / struct_conn / struct_ref / struct_ref_seq
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.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.name / _em_entity_assembly.entity_id_list / _entity_src_gen.pdbx_gene_src_gene / _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession
Description: Ligand identity / Details: Replacing Mg2+ by Mn2+ / Provider: author / Type: Coordinate replacement

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3
B: OST4 isoform 1
C: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST5
D: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST2
E: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1
F: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2
G: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit WBP1
H: OST3 isoform 1
P: PEPTIDE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)299,47829
Polymers287,8309
Non-polymers11,64820
Water1629
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area45860 Å2
ΔGint-310 kcal/mol
Surface area92520 Å2
MethodPISA

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Components

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Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ... , 6 types, 6 molecules ACDEFG

#1: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3 / Oligosaccharyl transferase subunit STT3 / Staurosporine and temperature sensitivity protein 3


Mass: 81604.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: STT3, YGL022W / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P39007, 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 genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: OST5, YGL226C-A, YGL226BC / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q92316
#4: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST2 / Oligosaccharyl transferase subunit OST2


Mass: 14712.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: OST2, GI527_G0005476 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H4BUV6
#5: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1


Mass: 54116.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: OST1, GI527_G0003255 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5PXA1
#6: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2


Mass: 31340.443 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SWP1, GI527_G0004591 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6V8S2Y6
#7: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit WBP1 / Oligosaccharyl transferase subunit WBP1


Mass: 49444.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: WBP1, GI527_G0001785 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H8ULL1

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Protein , 2 types, 2 molecules BH

#2: Protein OST4 isoform 1


Mass: 6871.818 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: OST4, GI527_G0000704 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A8H8UM72
#8: Protein OST3 isoform 1


Mass: 39518.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: OST3, GI527_G0005459 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q3S9

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Protein/peptide , 1 types, 1 molecules P

#9: Protein/peptide PEPTIDE


Mass: 696.707 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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Sugars , 3 types, 5 molecules

#10: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-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
DManpa1-2DManpa1-2DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,7,6/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3-3/a4-b1_b4-c1_c3-d1_c6-g1_d2-e1_e2-f1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#11: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#16: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / 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 , 6 types, 24 molecules

#12: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#13: Chemical
ChemComp-CPL / 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / PALMITOYL-LINOLEOYL PHOSPHATIDYLCHOLINE


Mass: 758.060 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C42H80NO8P / Comment: phospholipid*YM
#14: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#15: Chemical
ChemComp-KZB / (2~{S},3~{R},4~{R},5~{S},6~{S})-2-(hydroxymethyl)-6-[(1~{S},2~{R},3~{R},4~{R},5'~{S},6~{S},7~{R},8~{S},9~{R},12~{R},13~{R},15~{S},16~{S},18~{R})-5',7,9,13-tetramethyl-3,15-bis(oxidanyl)spiro[5-oxapentacyclo[10.8.0.0^{2,9}.0^{4,8}.0^{13,18}]icosane-6,2'-oxane]-16-yl]oxy-oxane-3,4,5-triol


Mass: 610.776 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C33H54O10
#17: Chemical ChemComp-323 / 2-[3,6-bis(dimethylamino)xanthen-9-yl]-5-methanoyl-benzoate / 5-Carboxy-N,N'-tetramethyl rhodamine


Mass: 414.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H22N2O4
#18: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: Yeast OST complex with acceptor peptide bound / Type: COMPLEX / Entity ID: #2-#5, #7-#8 / Source: NATURAL
Molecular weightValue: 0.284 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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 FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 48.4 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 72993 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00318076
ELECTRON MICROSCOPYf_angle_d0.67924646
ELECTRON MICROSCOPYf_dihedral_angle_d22.3236408
ELECTRON MICROSCOPYf_chiral_restr0.0452872
ELECTRON MICROSCOPYf_plane_restr0.0052958

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