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- EMDB-10257: Cryo-EM structure of yeast ALG6 in complex with 6AG9 Fab and Dol2... -

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Basic information

Entry
Database: EMDB / ID: EMD-10257
TitleCryo-EM structure of yeast ALG6 in complex with 6AG9 Fab and Dol25-P-Glc
Map dataYeast ALG6 in complex with 6AG9 Fab and Dol25-P-Glc
Sample
  • Complex: Nanodisc reconstituted yeast ALG6 in complex with 6AG9 Fab
    • Complex: ALG6
      • Protein or peptide: Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase
    • Complex: 6AG9 Fab
      • Protein or peptide: 6AG9 Fab heavy chain
      • Protein or peptide: 6AG9 Fab light chain
  • Ligand: glucosyl-dolichol phosphate
  • Ligand: water
KeywordsGlycosyltransferase / Glucosyltransferase / GT-C / N-Glycosylation / MEMBRANE PROTEIN
Function / homology
Function and homology information


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 / hexosyltransferase activity / protein N-linked glycosylation / protein glycosylation / aerobic respiration / endoplasmic reticulum membrane / endoplasmic reticulum
Similarity search - Function
Glycosyl transferase, ALG6/ALG8 / ALG6, ALG8 glycosyltransferase family
Similarity search - Domain/homology
Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsBloch JS / Pesciullesi G
Funding support Switzerland, 3 items
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, 2019-
Header (metadata) releaseSep 4, 2019-
Map releaseMar 11, 2020-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6snh
  • Surface level: 0.011
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10257.png

Downloads & links

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Map

FileDownload / File: emd_10257.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast ALG6 in complex with 6AG9 Fab and Dol25-P-Glc
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 300 pix.
= 252. Å		0.84 Å/pix.
x 300 pix.
= 252. Å		0.84 Å/pix.
x 300 pix.
= 252. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00747 / Movie #1: 0.013
Minimum - Maximum-0.017955348 - 0.039988324
Average (Standard dev.)0.00024831857 (±0.0015231628)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 251.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0180.0400.000

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Supplemental data

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Sample components

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Entire : Nanodisc reconstituted yeast ALG6 in complex with 6AG9 Fab

EntireName: Nanodisc reconstituted yeast ALG6 in complex with 6AG9 Fab
Components
  • Complex: Nanodisc reconstituted yeast ALG6 in complex with 6AG9 Fab
    • Complex: ALG6
      • Protein or peptide: Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase
    • Complex: 6AG9 Fab
      • Protein or peptide: 6AG9 Fab heavy chain
      • Protein or peptide: 6AG9 Fab light chain
  • Ligand: glucosyl-dolichol phosphate
  • Ligand: water

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Supramolecule #1: Nanodisc reconstituted yeast ALG6 in complex with 6AG9 Fab

SupramoleculeName: Nanodisc reconstituted yeast ALG6 in complex with 6AG9 Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 112.89472 KDa

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Supramolecule #2: ALG6

SupramoleculeName: ALG6 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: 6AG9 Fab

SupramoleculeName: 6AG9 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase

MacromoleculeName: Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: dolichyl-P-Glc:Man9GlcNAc2-PP-dolichol alpha-1,3-glucosyltransferase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 64.423223 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GPGGSTGRLA GAGGEFVDMA IGKRLLVNKP AEESFYASPM YDFLYPFRPV GNQWLPEYII FVCAVILRCT IGLGPYSGKG SPPLYGDFE AQRHWMEITQ HLPLSKWYWY DLQYWGLDYP PLTAFHSYLL GLIGSFFNPS WFALEKSRGF ESPDNGLKTY M RSTVIISD ...String:
GPGGSTGRLA GAGGEFVDMA IGKRLLVNKP AEESFYASPM YDFLYPFRPV GNQWLPEYII FVCAVILRCT IGLGPYSGKG SPPLYGDFE AQRHWMEITQ HLPLSKWYWY DLQYWGLDYP PLTAFHSYLL GLIGSFFNPS WFALEKSRGF ESPDNGLKTY M RSTVIISD ILFYFPAVIY FTKWLGRYRN QSPIGQSIAA SAILFQPSLM LIDHGHFQYN SVMLGLTAYA INNLLDEYYA MA AVCFVLS ICFKQMALYY APIFFAYLLS RSLLFPKFNI ARLTVIAFAT LATFAIIFAP LYFLGGGLKN IHQCIHRIFP FAR GIFEDK VANFWCVTNV FVKYKERFTI QQLQLYSLIA TVIGFLPAMI MTLLHPKKHL LPYVLIACSM SFFLFSFQVH EKTI LIPLL PITLLYSSTD WNVLSLVSWI NNVALFTLWP LLKKDGLHLQ YAVSFLLSNW LIGNFSFITP RFLPKSLTPG PSISS INSD YRRRSLLPYN VVWKSFIIGT YIAMGFYHFL DQFVAPPSKY PDLWVLLNCA VGFICFSIFW LWSYYKIFTS GSKSMK DL

UniProtKB: Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase

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Macromolecule #2: 6AG9 Fab heavy chain

MacromoleculeName: 6AG9 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 24.953664 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVYSSSIHWV RQAPGKGLEW VASISSYSGY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC AREYWSWYSY SYGIDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF P EPVTVSWN ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVYSSSIHWV RQAPGKGLEW VASISSYSGY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC AREYWSWYSY SYGIDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF P EPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHT

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Macromolecule #3: 6AG9 Fab light chain

MacromoleculeName: 6AG9 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.65025 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSYWVGYPI TFGQGTKVEI KRTVAAPSVF IFPPSDSQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSYWVGYPI TFGQGTKVEI KRTVAAPSVF IFPPSDSQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

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Macromolecule #4: glucosyl-dolichol phosphate

MacromoleculeName: glucosyl-dolichol phosphate / type: ligand / ID: 4 / Number of copies: 1 / Formula: LMH
Molecular weightTheoretical: 602.737 Da
Chemical component information

ChemComp-LMH:
glucosyl-dolichol phosphate

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 1 / 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

BufferpH: 7.4
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 2.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 115590
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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