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- PDB-9s6u: Ternary cryo-EM structure of human ALG9 with Dol25-PP-GlcNAc2Man8... -

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Basic information

Entry
Database: PDB / ID: 9s6u
TitleTernary cryo-EM structure of human ALG9 with Dol25-PP-GlcNAc2Man8, Dol25-P-Man and Fab
Components
  • Alpha-1,2-mannosyltransferase ALG9
  • Hs9-8 Fab heavy chain
  • Hs9-8 Fab light chain
KeywordsTRANSFERASE / Mannosyltransferase / ternary complex / N-linked glycosylation
Function / homology
Function and homology information


dolichyl-P-Man:Man6GlcNAc2-PP-dolichol alpha-1,2-mannosyltransferase / dolichyl-P-Man:Man8GlcNAc2-PP-dolichol alpha-1,2-mannosyltransferase / dol-P-Man:Man(8)GlcNAc(2)-PP-Dol alpha-1,2-mannosyltransferase activity / dol-P-Man:Man(6)GlcNAc(2)-PP-Dol alpha-1,2-mannosyltransferase activity / Defective ALG9 causes CDG-1l / alpha-1,2-mannosyltransferase activity / Biosynthesis of the N-glycan precursor (dolichol lipid-linked oligosaccharide, LLO) and transfer to a nascent protein / dolichol-linked oligosaccharide biosynthetic process / protein N-linked glycosylation / lumenal side of endoplasmic reticulum membrane ...dolichyl-P-Man:Man6GlcNAc2-PP-dolichol alpha-1,2-mannosyltransferase / dolichyl-P-Man:Man8GlcNAc2-PP-dolichol alpha-1,2-mannosyltransferase / dol-P-Man:Man(8)GlcNAc(2)-PP-Dol alpha-1,2-mannosyltransferase activity / dol-P-Man:Man(6)GlcNAc(2)-PP-Dol alpha-1,2-mannosyltransferase activity / Defective ALG9 causes CDG-1l / alpha-1,2-mannosyltransferase activity / Biosynthesis of the N-glycan precursor (dolichol lipid-linked oligosaccharide, LLO) and transfer to a nascent protein / dolichol-linked oligosaccharide biosynthetic process / protein N-linked glycosylation / lumenal side of endoplasmic reticulum membrane / endoplasmic reticulum membrane / membrane
Similarity search - Function
GPI mannosyltransferase / Alg9-like mannosyltransferase family
Similarity search - Domain/homology
: / : / CHOLESTEROL HEMISUCCINATE / Alpha-1,2-mannosyltransferase ALG9
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 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
Revision 1.0Mar 18, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
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
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.1Mar 25, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Alpha-1,2-mannosyltransferase ALG9
H: Hs9-8 Fab heavy chain
L: Hs9-8 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,4299
Polymers119,2453
Non-polymers4,1846
Water36020
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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Antibody , 2 types, 2 molecules HL

#2: Antibody Hs9-8 Fab heavy chain


Mass: 25638.363 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 Hs9-8 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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Protein / Sugars , 2 types, 2 molecules A

#1: Protein Alpha-1,2-mannosyltransferase ALG9 / Asparagine-linked glycosylation protein 9 homolog / Disrupted in bipolar disorder protein 1 / Dol-P- ...Asparagine-linked glycosylation protein 9 homolog / Disrupted in bipolar disorder protein 1 / Dol-P-Man:Man(6)GlcNAc(2)-PP-Dol alpha-1 / 2-mannosyltransferase / Dol-P-Man:Man(8)GlcNAc(2)-PP-Dol alpha-1


Mass: 70347.547 Da / Num. of mol.: 1 / Mutation: A82A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ALG9, DIBD1 / Production host: Homo sapiens (human)
References: UniProt: Q9H6U8, dolichyl-P-Man:Man6GlcNAc2-PP-dolichol alpha-1,2-mannosyltransferase, dolichyl-P-Man:Man8GlcNAc2-PP-dolichol alpha-1,2-mannosyltransferase
#4: 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.: 1 / 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, 25 molecules

#5: Chemical ChemComp-A1JMT / [(3S,6Z,10E,14E)-3,7,11,15,19-pentamethylicosa-6,10,14,18-tetraenyl] dihydrogen phosphate


Mass: 440.596 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H45O4P / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-A1JMB / [(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~{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]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: 2061.945 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C83H142N2O52P2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C31H50O4
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 20 / 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 human ALG9 with Hs9-8 Fab, anti-kappa light chain nanobody as well as Dol25-P-Man and Dol25-PP-GlcNAc2Man8 substratesCOMPLEX#1-#30MULTIPLE SOURCES
2Anti-kapp light chain nanobodyCOMPLEX1RECOMBINANT
3Hs9-8 FabCOMPLEX#2-#31RECOMBINANT
4ALG9COMPLEX#11RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Lama glama (llama)9844
43synthetic construct (others)32630
54Homo sapiens (human)9606
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.: 1.6 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: 2200 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 48 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: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 199158 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 53.7 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00226730
ELECTRON MICROSCOPYf_angle_d0.48579204
ELECTRON MICROSCOPYf_chiral_restr0.04061029
ELECTRON MICROSCOPYf_plane_restr0.00391105
ELECTRON MICROSCOPYf_dihedral_angle_d8.1281268

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