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- PDB-7oci: Cryo-EM structure of yeast Ost6p containing oligosaccharyltransfe... -

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Basic information

Entry
Database: PDB / ID: 7oci
TitleCryo-EM structure of yeast Ost6p containing oligosaccharyltransferase complex
Components(Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ...) x 9
KeywordsMEMBRANE PROTEIN / N-glycosylation / Yeast / Complex / Endoplasmic reticulum
Function / homology
Function and homology information


Miscellaneous transport and binding events / oligosaccharyltransferase I complex / oligosaccharyltransferase complex / dolichyl-diphosphooligosaccharide-protein glycotransferase / dolichyl-diphosphooligosaccharide-protein glycotransferase activity / protein N-linked glycosylation via asparagine / protein N-linked glycosylation / glycosyltransferase activity / protein-disulfide reductase activity / Neutrophil degranulation ...Miscellaneous transport and binding events / oligosaccharyltransferase I complex / oligosaccharyltransferase complex / dolichyl-diphosphooligosaccharide-protein glycotransferase / dolichyl-diphosphooligosaccharide-protein glycotransferase activity / protein N-linked glycosylation via asparagine / protein N-linked glycosylation / glycosyltransferase activity / 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 / 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
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Dolichylphosphate / 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 SWP1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST6 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST5 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsWild, R. / Neuhaus, J.D. / Eyring, J. / Irobalieva, R.N. / Kowal, J. / Lin, C.W. / Locher, K.P. / Aebi, M.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII5_173709 Switzerland
CitationJournal: Glycobiology / Year: 2021
Title: Functional analysis of Ost3p and Ost6p containing yeast oligosaccharyltransferases.
Abstract: The oligosaccharyltransferase (OST) is the central enzyme in the N-glycosylation pathway. It transfers a defined oligosaccharide from a lipid-linker onto the asparagine side chain of proteins. The ...The oligosaccharyltransferase (OST) is the central enzyme in the N-glycosylation pathway. It transfers a defined oligosaccharide from a lipid-linker onto the asparagine side chain of proteins. The yeast OST consists of eight subunits and exists in two catalytically distinct isoforms that differ in one subunit, Ost3p or Ost6p. The cryo-electron microscopy structure of the Ost6p containing complex was found to be highly similar to the Ost3p containing OST. OST enzymes with altered Ost3p/Ost6p subunits were generated and functionally analyzed. The three C-terminal transmembrane helices were responsible for the higher turnover-rate of the Ost3p vs. the Ost6p containing enzyme in vitro and the more severe hypoglycosylation in Ost3p lacking strains in vivo. Glycosylation of specific OST target sites required the N-terminal thioredoxin domain of Ost3p or Ost6p. This Ost3p/Ost6p dependence was glycosylation site but not protein specific. We concluded that the Ost3p/Ost6p subunits modulate the catalytic activity of OST and provide additional specificity for OST substrate recognition.
History
DepositionApr 27, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1
B: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST2
C: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST6
D: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST4
E: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST5
F: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3
G: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit WBP1
H: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit SWP1
I: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST6 - TM1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)295,17224
Polymers285,0549
Non-polymers10,11815
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area42590 Å2
ΔGint-317 kcal/mol
Surface area91450 Å2
MethodPISA

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Components

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Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ... , 9 types, 9 molecules ABCDEFGHI

#1: 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 S288C (yeast)
References: UniProt: P41543, dolichyl-diphosphooligosaccharide-protein glycotransferase
#2: 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 S288C (yeast)
References: UniProt: P46964, dolichyl-diphosphooligosaccharide-protein glycotransferase
#3: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST6 / Oligosaccharyl transferase subunit OST6 / Oligosaccharyl transferase 37 kDa subunit / OTase 37 kDa subunit


Mass: 37921.344 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q03723
#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 S288C (yeast)
References: UniProt: Q99380, dolichyl-diphosphooligosaccharide-protein glycotransferase
#5: 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 S288C (yeast)
References: UniProt: Q92316, dolichyl-diphosphooligosaccharide-protein glycotransferase
#6: 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 S288C (yeast)
References: UniProt: P39007, dolichyl-diphosphooligosaccharide-protein glycotransferase
#7: 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 S288C (yeast)
References: UniProt: P33767, dolichyl-diphosphooligosaccharide-protein glycotransferase
#8: 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 S288C (yeast)
References: UniProt: Q02795, dolichyl-diphosphooligosaccharide-protein glycotransferase
#9: Protein/peptide Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit OST6 - TM1


Mass: 2060.531 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: alpha-helix modelled as poly-UNK / Source: (natural) Saccharomyces cerevisiae S288C (yeast)
References: dolichyl-diphosphooligosaccharide-protein glycotransferase

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Sugars , 3 types, 5 molecules

#10: 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.: 1 / Source method: obtained synthetically
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#11: 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-Glycam 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
[]{[(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]{}}}}}LINUCSPDB-CARE
#13: 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.: 3 / 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 , 4 types, 10 molecules

#12: Chemical
ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#14: Chemical ChemComp-AJP / Digitonin


Mass: 1229.312 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C56H92O29 / Comment: detergent*YM
#15: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#16: Chemical ChemComp-V8K / Dolichylphosphate


Mass: 847.282 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C55H91O4P

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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

ComponentName: Ost6p containing yeast oligosaccharyltransferase complex
Type: COMPLEX / Entity ID: #2-#9 / Source: NATURAL
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Buffer solutionpH: 7.5
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 / C2 aperture diameter: 2.7 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 67 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategory
4RELIONCTF correction
7UCSF Chimeramodel fitting
10RELIONfinal Euler assignment
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220536 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6EZN

6ezn

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00317526
ELECTRON MICROSCOPYf_angle_d0.63923801
ELECTRON MICROSCOPYf_dihedral_angle_d13.816186
ELECTRON MICROSCOPYf_chiral_restr0.0482714
ELECTRON MICROSCOPYf_plane_restr0.0052920

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