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- PDB-6snh: Cryo-EM structure of yeast ALG6 in complex with 6AG9 Fab and Dol2... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6snh | ||||||||||||
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Title | Cryo-EM structure of yeast ALG6 in complex with 6AG9 Fab and Dol25-P-Glc | ||||||||||||
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![]() | MEMBRANE PROTEIN / Glycosyltransferase / Glucosyltransferase / GT-C / N-Glycosylation | ||||||||||||
Function / homology | ![]() dolichyl-P-Glc:Man9GlcNAc2-PP-dolichol alpha-1,3-glucosyltransferase / dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase activity / Biosynthesis of the N-glycan precursor (dolichol lipid-linked oligosaccharide, LLO) and transfer to a nascent protein / dolichol-linked oligosaccharide biosynthetic process / hexosyltransferase activity / protein N-linked glycosylation / protein glycosylation / aerobic respiration / endoplasmic reticulum membrane / endoplasmic reticulum Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() synthetic construct (others) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||||||||
![]() | Bloch, J.S. / Pesciullesi, G. / Boilevin, J. / Nosol, K. / Irobalieva, R.N. / Darbre, T. / Aebi, M. / Kossiakoff, A.A. / Reymond, J.L. / Locher, K.P. | ||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Structure and mechanism of the ER-based glucosyltransferase ALG6. Authors: Joël S Bloch / Giorgio Pesciullesi / Jérémy Boilevin / Kamil Nosol / Rossitza N Irobalieva / Tamis Darbre / Markus Aebi / Anthony A Kossiakoff / Jean-Louis Reymond / Kaspar P Locher / ![]() ![]() Abstract: In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins. The ...In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms. | ||||||||||||
History |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 205.2 KB | Display | ![]() |
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PDB format | ![]() | 148.4 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 956.9 KB | Display | ![]() |
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Full document | ![]() | 980.5 KB | Display | |
Data in XML | ![]() | 38.7 KB | Display | |
Data in CIF | ![]() | 58.2 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 10257MC ![]() 6sniC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 64423.223 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: ALG6, YOR002W, UNA544 / Plasmid: pOET1 / Production host: ![]() ![]() References: UniProt: Q12001, dolichyl-P-Glc:Man9GlcNAc2-PP-dolichol alpha-1,3-glucosyltransferase |
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#2: Antibody | Mass: 24953.664 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) Production host: ![]() ![]() |
#3: Antibody | Mass: 23650.250 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) Production host: ![]() ![]() |
#4: Chemical | ChemComp-LMH / |
#5: Water | ChemComp-HOH / |
Has ligand of interest | Y |
Has protein modification | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Molecular weight | Value: 0.11289472 MDa / Experimental value: NO | ||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.4 | ||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE-PROPANE |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 2.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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Processing
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115590 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 48.44 Å2 | ||||||||||||||||||||||||
Refine LS restraints |
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