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- PDB-6s7o: Cryo-EM structure of human oligosaccharyltransferase complex OST-A -

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Basic information

Entry
Database: PDB / ID: 6s7o
TitleCryo-EM structure of human oligosaccharyltransferase complex OST-A
Components
  • (Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ...) x 5
  • Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit
  • Oligosaccharyltransferase complex subunit OSTCOligosaccharyltransferase
  • Transmembrane protein 258Transmembrane protein
KeywordsTRANSFERASE / N-glycosylation / Oligosaccharyltransferase / OSTA
Function / homology
Function and homology information


oligosaccharyltransferase complex binding / oligosaccharyltransferase I complex / oligosaccharyltransferase III complex / Asparagine N-linked glycosylation / membrane-bounded organelle / co-translational protein modification / oligosaccharyltransferase complex / dolichyl-diphosphooligosaccharide-protein glycotransferase / dolichyl-diphosphooligosaccharide-protein glycotransferase activity / protein N-linked glycosylation via asparagine ...oligosaccharyltransferase complex binding / oligosaccharyltransferase I complex / oligosaccharyltransferase III complex / Asparagine N-linked glycosylation / membrane-bounded organelle / co-translational protein modification / oligosaccharyltransferase complex / dolichyl-diphosphooligosaccharide-protein glycotransferase / dolichyl-diphosphooligosaccharide-protein glycotransferase activity / protein N-linked glycosylation via asparagine / protein N-linked glycosylation / epithelial cell apoptotic process / azurophil granule membrane / protein glycosylation / blastocyst development / Advanced glycosylation endproduct receptor signaling / SRP-dependent cotranslational protein targeting to membrane / enzyme activator activity / rough endoplasmic reticulum / response to endoplasmic reticulum stress / T cell activation / post-translational protein modification / response to cytokine / protein modification process / regulation of protein stability / melanosome / transferase activity / Maturation of spike protein / nuclear body / inflammatory response / intracellular membrane-bounded organelle / apoptotic process / Neutrophil degranulation / endoplasmic reticulum membrane / negative regulation of apoptotic process / endoplasmic reticulum / protein-containing complex / RNA binding / membrane / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
DAD/Ost2 / Oligosaccharyltransferase complex subunit / DAD family / Oligosaccharyltransferase subunit 5 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48kDa subunit / Oligosaccharyltransferase complex subunit OSTC / Oligosaccharyltransferase 48 kDa subunit beta / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit Swp1 / Oligosaccharyltransferase subunit Ribophorin II / Oligosaccaryltransferase ...DAD/Ost2 / Oligosaccharyltransferase complex subunit / DAD family / Oligosaccharyltransferase subunit 5 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48kDa subunit / Oligosaccharyltransferase complex subunit OSTC / 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 / Oligosaccharyl transferase, STT3 subunit / Oligosaccharyl transferase STT3, N-terminal
Similarity search - Domain/homology
Chem-EGY / Chem-KZB / Chem-KZE / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A / Transmembrane protein 258 ...Chem-EGY / Chem-KZB / Chem-KZE / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A / Transmembrane protein 258 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1 / Oligosaccharyltransferase complex subunit OSTC
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRamirez, A.S. / Kowal, J. / Locher, K.P.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII3_147632 Switzerland
CitationJournal: Science / Year: 2019
Title: Cryo-electron microscopy structures of human oligosaccharyltransferase complexes OST-A and OST-B.
Authors: Ana S Ramírez / Julia Kowal / Kaspar P Locher /
Abstract: Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum. Mammals express two distinct OST complexes that act in a ...Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum. Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner. Here, we present high-resolution cryo-electron microscopy structures of human OST-A and OST-B. Although they have similar overall architectures, structural differences in the catalytic subunits STT3A and STT3B facilitate contacts to distinct OST subunits, DC2 in OST-A and MAGT1 in OST-B. In OST-A, interactions with TMEM258 and STT3A allow ribophorin-I to form a four-helix bundle that can bind to a translating ribosome, whereas the equivalent region is disordered in OST-B. We observed an acceptor peptide and dolichylphosphate bound to STT3B, but only dolichylphosphate in STT3A, suggesting distinct affinities of the two OST complexes for protein substrates.
History
DepositionJul 5, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 18, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2019Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
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_unobs_or_zero_occ_atoms / 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.type_symbol / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_unobs_or_zero_occ_atoms.auth_asym_id / _pdbx_unobs_or_zero_occ_atoms.auth_seq_id / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _pdbx_validate_close_contact.auth_asym_id_1 / _pdbx_validate_close_contact.auth_asym_id_2 / _pdbx_validate_close_contact.auth_seq_id_1 / _pdbx_validate_close_contact.auth_seq_id_2 / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_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 STT3A
B: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4
C: Transmembrane protein 258
D: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1
E: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1
F: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2
G: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit
H: Oligosaccharyltransferase complex subunit OSTC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)325,88630
Polymers311,9938
Non-polymers13,89322
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area41470 Å2
ΔGint-243 kcal/mol
Surface area100010 Å2
MethodPISA

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Components

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Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ... , 5 types, 5 molecules ABDEF

#1: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A / STT3-A / B5 / Integral membrane protein 1 / Transmembrane protein TMC


Mass: 80607.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
References: UniProt: P46977, dolichyl-diphosphooligosaccharide-protein glycotransferase
#2: Protein/peptide Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4


Mass: 4196.004 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P0C6T2
#4: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1 / Oligosaccharyl transferase subunit DAD1 / Defender against cell death 1 / DAD-1


Mass: 12503.631 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P61803
#5: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 67 kDa subunit / Ribophorin I / RPN- ...Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 67 kDa subunit / Ribophorin I / RPN-I / Ribophorin-1


Mass: 68656.156 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P04843
#6: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 63 kDa subunit / RIBIIR / Ribophorin ...Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 63 kDa subunit / RIBIIR / Ribophorin II / RPN-II / Ribophorin-2


Mass: 69347.508 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P04844

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Protein , 3 types, 3 molecules CGH

#3: Protein Transmembrane protein 258 / Transmembrane protein / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit TMEM258 / Oligosaccharyl ...Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit TMEM258 / Oligosaccharyl transferase subunit TMEM258


Mass: 9083.804 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P61165
#7: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / Oligosaccharyl transferase 48 kDa subunit


Mass: 50754.438 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: A0A024RAD5, UniProt: P39656*PLUS
#8: Protein Oligosaccharyltransferase complex subunit OSTC / Oligosaccharyltransferase / Hydrophobic protein HSF-28


Mass: 16844.215 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: OSTC, DC2, HDCMD45P, HSPC307 / Production host: Homo sapiens (human) / References: UniProt: Q9NRP0

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Sugars , 3 types, 3 molecules

#9: 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-ROHGlycam 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
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#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)-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: 1397.245 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-2DManpa1-3[DManpa1-6DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,8,7/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3-3-3/a4-b1_b4-c1_c3-d1_c6-g1_d2-e1_e2-f1_g6-h1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-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]{[(6+1)][a-D-Manp]{}}}}}}LINUCSPDB-CARE
#11: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-3)-[alpha-D- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]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: 1397.245 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-3[DManpa1-3[DManpa1-6]DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,8,7/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3-3-3/a4-b1_b4-c1_c3-d1_c6-f1_d2-e1_f3-g1_f6-h1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}[(6+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}}LINUCSPDB-CARE

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Non-polymers , 4 types, 19 molecules

#12: 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.: 9
Source method: isolated from a genetically manipulated source
Formula: C33H54O10
#13: 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.: 7
Source method: isolated from a genetically manipulated source
Formula: C33H67NO8P / Comment: phospholipid*YM
#14: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: Mg
#15: Chemical ChemComp-KZE / [(3~{R},6~{Z},10~{Z},14~{Z},18~{Z})-3,7,11,15,19,23-hexamethyltetracosa-6,10,14,18,22-pentaenyl] dihydrogen phosphate


Mass: 508.713 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C30H53O4P

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Human oligosaccharyltransferase complex OST-ACOMPLEX#1-#80MULTIPLE SOURCES
2Human oligosaccharyltransferase complexOligosaccharyltransferaseCOMPLEX#1-#71NATURAL
3Oligosaccharyltransferase complex subunit OSTCOligosaccharyltransferaseCOMPLEX#81RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)Organism: Homo sapiens (human)
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 FIELDBright-field microscopy / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 68 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 6035
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4GctfCTF correction
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 424817
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 156950 / Symmetry type: POINT

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