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- PDB-9s6r: Ternary cryo-EM structure of yeast ALG3 with Dol25-PP-GlcNAc2Man5... -

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Basic information

Entry
Database: PDB / ID: 9s6r
TitleTernary cryo-EM structure of yeast ALG3 with Dol25-PP-GlcNAc2Man5, Dol25-P-Man, and Fab
Components
  • Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase
  • Sc-16 Fab heavy chain
  • Sc-16 Fab light chain
KeywordsTRANSFERASE / Mannosyltransferase / ternary complex / N-linked glycosylation
Function / homology
Function and homology information


dolichyl-P-Man:Man5GlcNAc2-PP-dolichol alpha-1,3-mannosyltransferase / dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase activity / Biosynthesis of the N-glycan precursor (dolichol lipid-linked oligosaccharide, LLO) and transfer to a nascent protein / dolichol-linked oligosaccharide biosynthetic process / endoplasmic reticulum membrane / endoplasmic reticulum
Similarity search - Function
Glycosyltransferase, ALG3 / ALG3 protein
Similarity search - Domain/homology
: / Chem-IZY / CHOLESTEROL HEMISUCCINATE / Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase
Similarity search - Component
Biological speciessynthetic construct (others)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsAlexander, J.A.N. / Chen, S.Y. / Mukherjee, S. / de Capitani, M. / Irobalieva, R.N. / Rossi, L. / Agrawal, P. / Kowal, J. / Meirelles, M.A. / Aebi, M. ...Alexander, J.A.N. / Chen, S.Y. / Mukherjee, S. / de Capitani, M. / Irobalieva, R.N. / Rossi, L. / Agrawal, P. / Kowal, J. / Meirelles, M.A. / Aebi, M. / Reymond, J.L. / Kossiakoff, A.A. / Riniker, S. / Locher, K.P.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_196862 Switzerland
Swiss National Science Foundation315230_220007 Switzerland
CitationJournal: Nat Chem Biol / Year: 2026
Title: Structures of ALG3/9/12 reveal the assembly logic of the N-glycan oligomannose core.
Authors: J Andrew N Alexander / Shu-Yu Chen / Somnath Mukherjee / Mario de Capitani / Rossitza N Irobalieva / Lorenzo Rossi / Parth Agrawal / Julia Kowal / Matheus A Meirelles / Markus Aebi / Jean- ...Authors: J Andrew N Alexander / Shu-Yu Chen / Somnath Mukherjee / Mario de Capitani / Rossitza N Irobalieva / Lorenzo Rossi / Parth Agrawal / Julia Kowal / Matheus A Meirelles / Markus Aebi / Jean-Louis Reymond / Anthony A Kossiakoff / Sereina Riniker / Kaspar P Locher /
Abstract: Asparagine-linked glycans are essential for the maturation and function of most eukaryotic secretory proteins. The biosynthesis and transfer of dolichylpyrophosphate-anchored GlcNAcManGlc glycan is a ...Asparagine-linked glycans are essential for the maturation and function of most eukaryotic secretory proteins. The biosynthesis and transfer of dolichylpyrophosphate-anchored GlcNAcManGlc glycan is a highly conserved process occurring in the endoplasmic reticulum (ER) membrane and involving over a dozen membrane proteins whose dysfunction is linked to congenital disorders of glycosylation (CDGs). Three membrane-integral mannosyltransferases, ALG3, ALG9 and ALG12, mediate four consecutive mannosylation reactions that convert GlcNAcMan to GlcNAcMan. Here, using chemoenzymatically synthesized lipid-linked glycan donor and acceptor analogs, we recapitulated this biosynthetic pathway in vitro. High-resolution cryo-electron microscopy structures of pseudo-Michaelis complexes of each step revealed how the branched glycan is accurately synthesized and unwanted side products are averted. Molecular dynamics simulations and mutagenesis studies uncovered a subtle but precise mechanism selecting the dolichylphosphomannose donor substrate over dolichylphosphoglucose, which is also present in the ER membrane. Our results also provide mechanistic explanations for enzyme dysfunction in CDGs and offer opportunities for N-glycan engineering.
History
DepositionAug 1, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 18, 2026Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Mar 18, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 25, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Sc-16 Fab heavy chain
L: Sc-16 Fab light chain
A: Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)105,5646
Polymers102,8993
Non-polymers2,6653
Water18010
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 1 types, 1 molecules A

#3: Protein Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase / Asparagine-linked glycosylation protein 3 / Dol-P-Man-dependent alpha(1-3)-mannosyltransferase / ...Asparagine-linked glycosylation protein 3 / Dol-P-Man-dependent alpha(1-3)-mannosyltransferase / Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase / Dolichyl-phosphate-mannose--glycolipid alpha-mannosyltransferase / HM-1 killer toxin resistance protein


Mass: 53925.871 Da / Num. of mol.: 1 / Mutation: D71N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ALG3, RHK1, YBL082C, YBL0720 / Production host: Homo sapiens (human)
References: UniProt: P38179, dolichyl-P-Man:Man5GlcNAc2-PP-dolichol alpha-1,3-mannosyltransferase

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Antibody , 2 types, 2 molecules HL

#1: Antibody Sc-16 Fab heavy chain


Mass: 25714.615 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)
#2: Antibody Sc-16 Fab light chain


Mass: 23258.783 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)

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Non-polymers , 4 types, 13 molecules

#4: Chemical ChemComp-A1JMA / [(2~{R},3~{R},4~{R},5~{S},6~{R})-3-acetamido-5-[(2~{S},3~{R},4~{R},5~{S},6~{R})-3-acetamido-6-(hydroxymethyl)-5-[(2~{S},3~{S},4~{S},5~{R},6~{R})-4-[(2~{R},3~{S},4~{S},5~{S},6~{R})-6-(hydroxymethyl)-3-[(2~{S},3~{S},4~{S},5~{S},6~{R})-6-(hydroxymethyl)-3,4,5-tris(oxidanyl)oxan-2-yl]oxy-4,5-bis(oxidanyl)oxan-2-yl]oxy-6-[[(2~{S},3~{S},4~{S},5~{S},6~{R})-6-(hydroxymethyl)-3,4,5-tris(oxidanyl)oxan-2-yl]oxymethyl]-3,5-bis(oxidanyl)oxan-2-yl]oxy-4-oxidanyl-oxan-2-yl]oxy-6-(hydroxymethyl)-4-oxidanyl-oxan-2-yl] [oxidanyl-[(3~{S},6~{Z},10~{E},14~{E})-3,7,11,15,19-pentamethylicosa-6,10,14,18-tetraenoxy]phosphoryl] hydrogen phosphate


Mass: 1575.524 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C65H112N2O37P2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#6: Chemical ChemComp-IZY / Dolichol monophosphate beta-D-Mannose / [(2~{S},3~{S},4~{S},5~{S},6~{R})-6-(hydroxymethyl)-3,4,5-tris(oxidanyl)oxan-2-yl] [(3~{S},6~{Z},10~{E},14~{E})-3,7,11,15,19-pentamethylicosa-6,10,14,18-tetraenyl] hydrogen phosphate


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


Mass: 18.015 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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
1Ternary complex of yeast ALG3 with Sc3-16 Fab, anti-kappa light chain nanobody as well as Dol25-P-Man and Dol25-PP-GlcNAc2Man5 substratesCOMPLEX#1-#30MULTIPLE SOURCES
2Anti-kapp light chain nanobodyCOMPLEX#21RECOMBINANT
3Sc3-16 FabCOMPLEX#1-#21RECOMBINANT
4ALG3COMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.12 MDaNO
21NO
31NO
43
54
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Lama glama (llama)9844
43synthetic construct (others)32630
54Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
32Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
43Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
54Homo sapiens (human)9606
Buffer solutionpH: 7.5
SpecimenConc.: 2.4 mg/ml / Embedding 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2800 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 63 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.2_5419model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 243185 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 57.39 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035252
ELECTRON MICROSCOPYf_angle_d0.52277161
ELECTRON MICROSCOPYf_chiral_restr0.0406825
ELECTRON MICROSCOPYf_plane_restr0.0035850
ELECTRON MICROSCOPYf_dihedral_angle_d8.2962907

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