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- EMDB-54632: Ternary cryo-EM structure of human ALG9 with Dol25-PP-GlcNAc2Man8... -

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Entry
Database: EMDB / ID: EMD-54632
TitleTernary cryo-EM structure of human ALG9 with Dol25-PP-GlcNAc2Man8, Dol25-P-Man and Fab
Map data
Sample
  • Complex: Ternary complex of human ALG9 with Hs9-8 Fab, anti-kappa light chain nanobody as well as Dol25-P-Man and Dol25-PP-GlcNAc2Man8 substrates
    • Complex: Anti-kapp light chain nanobody
    • Complex: Hs9-8 Fab
      • Protein or peptide: Hs9-8 Fab heavy chain
      • Protein or peptide: Hs9-8 Fab light chain
    • Complex: ALG9
      • Protein or peptide: Alpha-1,2-mannosyltransferase ALG9
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: [(3S,6Z,10E,14E)-3,7,11,15,19-pentamethylicosa-6,10,14,18-tetraenyl] dihydrogen phosphate
  • Ligand: [(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
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water
KeywordsMannosyltransferase / ternary complex / N-linked glycosylation / TRANSFERASE
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
Alpha-1,2-mannosyltransferase ALG9
Similarity search - Component
Biological speciesLama glama (llama) / synthetic construct (others) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsAlexander JAN / Chen SY / Mukherjee S / de Capitani M / Irobalieva RN / Rossi L / Agrawal P / Kowal J / Meirelles MA / Aebi M ...Alexander JAN / Chen SY / Mukherjee S / de Capitani M / Irobalieva RN / Rossi L / Agrawal P / Kowal J / Meirelles MA / Aebi M / Reymond JL / Kossiakoff AA / Riniker S / Locher KP
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_196862 Switzerland
Swiss National Science Foundation315230_220007 Switzerland
CitationJournal: Nat.Chem.Biol. / Year: 2026
Title: Structural basis for oligomannose N-glycan synthesis in the endoplasmic reticulum
Authors: Alexander JAN / Chen SY / Mukherjee S / de Capitani M / Irobalieva RN / Rossi L / Agrawal P / Kowal J / Meirelles MA / Aebi M / Reymond JL / Kossiakoff AA / Riniker S / Locher KP
History
DepositionAug 1, 2025-
Header (metadata) releaseMar 18, 2026-
Map releaseMar 18, 2026-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_54632.png

Downloads & links

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Map

FileDownload / File: emd_54632.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 432 pix.
= 279.936 Å		0.65 Å/pix.
x 432 pix.
= 279.936 Å		0.65 Å/pix.
x 432 pix.
= 279.936 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.648 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.4766418 - 0.7035373
Average (Standard dev.)0.00010010608 (±0.013229684)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 279.936 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_54632_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_54632_half_map_2.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of human ALG9 with Hs9-8 Fab, anti-kappa light ch...

EntireName: Ternary complex of human ALG9 with Hs9-8 Fab, anti-kappa light chain nanobody as well as Dol25-P-Man and Dol25-PP-GlcNAc2Man8 substrates
Components
  • Complex: Ternary complex of human ALG9 with Hs9-8 Fab, anti-kappa light chain nanobody as well as Dol25-P-Man and Dol25-PP-GlcNAc2Man8 substrates
    • Complex: Anti-kapp light chain nanobody
    • Complex: Hs9-8 Fab
      • Protein or peptide: Hs9-8 Fab heavy chain
      • Protein or peptide: Hs9-8 Fab light chain
    • Complex: ALG9
      • Protein or peptide: Alpha-1,2-mannosyltransferase ALG9
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: [(3S,6Z,10E,14E)-3,7,11,15,19-pentamethylicosa-6,10,14,18-tetraenyl] dihydrogen phosphate
  • Ligand: [(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
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water

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Supramolecule #1: Ternary complex of human ALG9 with Hs9-8 Fab, anti-kappa light ch...

SupramoleculeName: Ternary complex of human ALG9 with Hs9-8 Fab, anti-kappa light chain nanobody as well as Dol25-P-Man and Dol25-PP-GlcNAc2Man8 substrates
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 130 KDa

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Supramolecule #2: Anti-kapp light chain nanobody

SupramoleculeName: Anti-kapp light chain nanobody / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Lama glama (llama)

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Supramolecule #3: Hs9-8 Fab

SupramoleculeName: Hs9-8 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: synthetic construct (others)

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Supramolecule #4: ALG9

SupramoleculeName: ALG9 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Alpha-1,2-mannosyltransferase ALG9

MacromoleculeName: Alpha-1,2-mannosyltransferase ALG9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: dolichyl-P-Man:Man6GlcNAc2-PP-dolichol alpha-1,2-mannosyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 70.347547 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPGGSTMASR GARQRLKGSG ASSGDTAPAA DKLRELLGSR EAGGAEHRTE LSGNKAGQVW APEGSTAFKC LLSARLCAAL LSNISDCAE TFNYWEPTHY LIYGEGFQTW EYSPAYAIRS YAYLLLHAWP AAFHARILQT NKILVFYFLR CLLAFVSCIC E LYFYKAVC ...String:
GPGGSTMASR GARQRLKGSG ASSGDTAPAA DKLRELLGSR EAGGAEHRTE LSGNKAGQVW APEGSTAFKC LLSARLCAAL LSNISDCAE TFNYWEPTHY LIYGEGFQTW EYSPAYAIRS YAYLLLHAWP AAFHARILQT NKILVFYFLR CLLAFVSCIC E LYFYKAVC KKFGLHVSRM MLAFLVLSTG MFCSSSAFLP SSFCMYTTLI AMTGWYMDKT SIAVLGVAAG AILGWPFSAA LG LPIAFDL LVMKHRWKSF FHWSLMALIL FLVPVVVIDS YYYGKLVIAP LNIVLYNVFT PHGPDLYGTE PWYFYLINGF LNF NVAFAL ALLVLPLTSL MEYLLQRFHV QNLGHPYWLT LAPMYIWFII FFIQPHKEER FLFPVYPLIC LCGAVALSAL QKCY HFVFQ RYRLEHYTVT SNWLALGTVF LFGLLSFSRS VALFRGYHGP LDLYPEFYRI ATDPTIHTVP EGRPVNVCVG KEWYR FPSS FLLPDNWQLQ FIPSEFRGQL PKPFAEGPLA TRIVPTDMND QNLEEPSRYI DISKCHYLVD LDTMRETPRE PKYSSN KEE WISLAYRPFL DASRSSKLLR AFYVPFLSDQ YTVYVNYTIL KPRKAKQIRK KSGG

UniProtKB: Alpha-1,2-mannosyltransferase ALG9

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Macromolecule #2: Hs9-8 Fab heavy chain

MacromoleculeName: Hs9-8 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.638363 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NIYYSSIHWV RQAPGKGLEW VASISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARYWDGWQYS DSWYYSPYAI DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NIYYSSIHWV RQAPGKGLEW VASISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARYWDGWQYS DSWYYSPYAI DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HT

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Macromolecule #3: Hs9-8 Fab light chain

MacromoleculeName: Hs9-8 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.258783 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: [(3S,6Z,10E,14E)-3,7,11,15,19-pentamethylicosa-6,10,14,18-tetraen...

MacromoleculeName: [(3S,6Z,10E,14E)-3,7,11,15,19-pentamethylicosa-6,10,14,18-tetraenyl] dihydrogen phosphate
type: ligand / ID: 5 / Number of copies: 1 / Formula: A1JMT
Molecular weightTheoretical: 440.596 Da

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Macromolecule #6: [(2~{R},3~{R},4~{R},5~{S},6~{R})-3-acetamido-5-[(2~{S},3~{R},4~{R...

MacromoleculeName: [(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- ...Name: [(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
type: ligand / ID: 6 / Number of copies: 1 / Formula: A1JMB
Molecular weightTheoretical: 2.061945 KDa

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Macromolecule #7: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 7 / Number of copies: 3 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 20 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.6 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 199158
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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