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- PDB-6snh: Cryo-EM structure of yeast ALG6 in complex with 6AG9 Fab and Dol2... -

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Basic information

Entry
Database: PDB / ID: 6snh
TitleCryo-EM structure of yeast ALG6 in complex with 6AG9 Fab and Dol25-P-Glc
Components
  • 6AG9 Fab heavy chain
  • 6AG9 Fab light chain
  • Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase
KeywordsMEMBRANE PROTEIN / Glycosyltransferase / Glucosyltransferase / GT-C / N-Glycosylation
Function / homology
Function and homology information


dolichyl-P-Glc:Man9GlcNAc2-PP-dolichol alpha-1,3-glucosyltransferase / oligosaccharide-lipid intermediate biosynthetic process / 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
Glycosyl transferase, ALG6/ALG8 / Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase / ALG6, ALG8 glycosyltransferase family
Similarity search - Domain/homology
glucosyl-dolichol phosphate / Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsBloch, 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 Switzerland, 3items
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII3_147632 Switzerland
Swiss National Science FoundationCRSII5_173709 Switzerland
Swiss National Science Foundation310030B_166672 Switzerland
CitationJournal: Nature / Year: 2020
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
DepositionAug 24, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 11, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 18, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 1, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
X: Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase
H: 6AG9 Fab heavy chain
L: 6AG9 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,6304
Polymers113,0273
Non-polymers6031
Water181
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5600 Å2
ΔGint-39 kcal/mol
Surface area40660 Å2
MethodPISA

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Components

#1: Protein Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase / Asparagine-linked glycosylation protein 6 / Dol-P-Glc:Man(9)GlcNAc(2)-PP-Dol alpha-1 / 3- ...Asparagine-linked glycosylation protein 6 / Dol-P-Glc:Man(9)GlcNAc(2)-PP-Dol alpha-1 / 3-glucosyltransferase / Dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl glucosyltransferase


Mass: 64423.223 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ALG6, YOR002W, UNA544 / Plasmid: pOET1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q12001, dolichyl-P-Glc:Man9GlcNAc2-PP-dolichol alpha-1,3-glucosyltransferase
#2: Antibody 6AG9 Fab heavy chain


Mass: 24953.664 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#3: Antibody 6AG9 Fab light chain


Mass: 23650.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#4: Chemical ChemComp-LMH / glucosyl-dolichol phosphate


Mass: 602.737 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H55O9P / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
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

Component
IDNameTypeEntity IDParent-IDSource
1Nanodisc reconstituted yeast ALG6 in complex with 6AG9 FabCOMPLEX#1-#30MULTIPLE SOURCES
2ALG6COMPLEX#11RECOMBINANT
36AG9 FabCOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.11289472 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (brewer's yeast)4932
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
Buffer solutionpH: 7.4
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
Image recordingElectron dose: 2.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassificationNB
PHENIX1.16_3549refinement
PHENIX1.16_3549refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115590 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 48.44 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00787545
ELECTRON MICROSCOPYf_angle_d0.912510292
ELECTRON MICROSCOPYf_chiral_restr0.06241141
ELECTRON MICROSCOPYf_plane_restr0.00471267
ELECTRON MICROSCOPYf_dihedral_angle_d12.77924392

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