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Yorodumi- PDB-6ezn: Cryo-EM structure of the yeast oligosaccharyltransferase (OST) complex -
+Open data
-Basic information
Entry | Database: PDB / ID: 6ezn | |||||||||
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Title | Cryo-EM structure of the yeast oligosaccharyltransferase (OST) complex | |||||||||
Components | (Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ...) x 8 | |||||||||
Keywords | MEMBRANE PROTEIN / OST complex / oligosaccharyltransferase / N-linked glycosylation / yeast | |||||||||
Function / homology | Function and homology information Miscellaneous transport and binding events / : / dolichol-linked oligosaccharide biosynthetic process / 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 ...Miscellaneous transport and binding events / : / dolichol-linked oligosaccharide biosynthetic process / 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 / nuclear envelope / protein-macromolecule adaptor activity / endoplasmic reticulum membrane / structural molecule activity / endoplasmic reticulum / mitochondrion / membrane / metal ion binding Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
Authors | Wild, R. / Kowal, J. / Eyring, J. / Ngwa, E.M. / Aebi, M. / Locher, K.P. | |||||||||
Funding support | Switzerland, 2items
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Citation | Journal: 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. #1: Journal: Science / Year: 2018 Title: Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation. Authors: Rebekka Wild / Julia Kowal / Jillianne Eyring / Elsy M Ngwa / Markus Aebi / Kaspar P Locher / Abstract: Oligosaccharyltransferase (OST) is an essential membrane protein complex in the endoplasmic reticulum, where it transfers an oligosaccharide from a dolichol-pyrophosphate-activated donor to ...Oligosaccharyltransferase (OST) is an essential membrane protein complex in the endoplasmic reticulum, where it transfers an oligosaccharide from a dolichol-pyrophosphate-activated donor to glycosylation sites of secretory proteins. Here we describe the atomic structure of yeast OST determined by cryo-electron microscopy, revealing a conserved subunit arrangement. The active site of the catalytic STT3 subunit points away from the center of the complex, allowing unhindered access to substrates. The dolichol-pyrophosphate moiety binds to a lipid-exposed groove of STT3, whereas two noncatalytic subunits and an ordered N-glycan form a membrane-proximal pocket for the oligosaccharide. The acceptor polypeptide site faces an oxidoreductase domain in stand-alone OST complexes or is immediately adjacent to the translocon, suggesting how eukaryotic OSTs efficiently glycosylate a large number of polypeptides before their folding. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6ezn.cif.gz | 390 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6ezn.ent.gz | 314.5 KB | Display | PDB format |
PDBx/mmJSON format | 6ezn.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6ezn_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 6ezn_full_validation.pdf.gz | 1.5 MB | Display | |
Data in XML | 6ezn_validation.xml.gz | 69 KB | Display | |
Data in CIF | 6ezn_validation.cif.gz | 100.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ez/6ezn ftp://data.pdbj.org/pub/pdb/validation_reports/ez/6ezn | HTTPS FTP |
-Related structure data
Related structure data | 4161MC 8agbC 8agcC 8ageC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ... , 8 types, 8 molecules ABCDEFGH
#1: Protein | 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 |
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#2: Protein | 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 |
#3: Protein | 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 |
#4: Protein/peptide | 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 | 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 |
#6: Protein | 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 |
#7: Protein | 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 |
#8: Protein | 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 |
-Sugars , 4 types, 5 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 Source method: isolated from a genetically manipulated source | ||||
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#10: Polysaccharide | Source method: isolated from a genetically manipulated source #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-(1-3)-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 | Source method: isolated from a genetically manipulated source #14: Sugar | ChemComp-NAG / | |
-Non-polymers , 2 types, 5 molecules
#12: Chemical | ChemComp-CPL / #13: Chemical | ChemComp-PTY / | |
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-Details
Compound details | Amino acid residues H22 to H65 in Swp1 were built as PolyA chain and renamed to specific amino ...Amino acid residues H22 to H65 in Swp1 were built as PolyA chain and renamed to specific amino acids according to residue numbers on request by wwPDB |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: yeast oligosaccharyltransferase (OST) complex / Type: COMPLEX / Entity ID: #1-#8 / Source: NATURAL |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Buffer solution | pH: 7.5 / Details: 20 mM HEPES, pH 7.5, 150 mM NaCl |
Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: OST complex reconstituted into nanodisc |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 95 % / Chamber temperature: 277 K Details: Quantifoil carbon grids (300 mesh, copper) were glow discharged at 25 mA for 40 s. 3.5 ul of nanodisc-reconstituted OST sample at concentration of 0.5 mg/ml was applied onto the grid and ...Details: Quantifoil carbon grids (300 mesh, copper) were glow discharged at 25 mA for 40 s. 3.5 ul of nanodisc-reconstituted OST sample at concentration of 0.5 mg/ml was applied onto the grid and grids were blotted for 3 s and flash-frozen in a mixture of liquid ethane and propane cooled by liquid nitrogen. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 0.25 sec. / Electron dose: 2 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3915 |
EM imaging optics | Energyfilter name: GIF Quantum LS |
-Processing
Software | Name: PHENIX / Version: 1.11.1_2575: / Classification: refinement | ||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 486361 | ||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110091 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL | ||||||||||||||||||||||||||||||||
Refine LS restraints |
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