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- EMDB-10258: Cryo-EM structure of nanodisc reconstituted yeast ALG6 in complex... -

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Basic information

Entry
Database: EMDB / ID: EMD-10258
TitleCryo-EM structure of nanodisc reconstituted yeast ALG6 in complex with 6AG9 Fab
Map dataNanodisc reconstituted yeast ALG6 in complex with 6AG9 Fab
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: PHOSPHATIDYLETHANOLAMINE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water
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
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: 3.0 Å
AuthorsBloch JS / Pesciullesi G / Boilevin J / Nosol K / Irobalieva RN / Darbre T / Aebi M / Kossiakoff AA / Reymond JL / Locher KP
Funding support Switzerland, 3 items
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII3_147632 Switzerland
Swiss National Science Foundation310030B_166672 Switzerland
Swiss National Science FoundationCRSII5_173709 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-
UpdateApr 1, 2020-
Current statusApr 1, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6sni
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10258.png

Downloads & links

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Map

FileDownload / File: emd_10258.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNanodisc reconstituted yeast ALG6 in complex with 6AG9 Fab
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.00768 / Movie #1: 0.019
Minimum - Maximum-0.09224564 - 0.14414388
Average (Standard dev.)-0.00001527076 (±0.0032000486)
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-0.0920.144-0.000

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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: PHOSPHATIDYLETHANOLAMINE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • 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)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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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)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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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

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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: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 4 / Number of copies: 5 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM / Phosphatidylethanolamine

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Macromolecule #5: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 5 / Number of copies: 3 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 7 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

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

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 171764
FSC plot (resolution estimation)

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