[English] 日本語
Yorodumi
- EMDB-10258: Cryo-EM structure of nanodisc reconstituted yeast ALG6 in complex... -

+
Open data


ID or keywords:

Loading...

-
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
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 / dolichol-linked oligosaccharide biosynthetic process / hexosyltransferase activity / : / 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: 3.0 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-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBe / Status: Released

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

emd_10258.png

Downloads & links

-
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
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: 0.00768 / Movie #1: 0.019
Minimum - Maximum-0.09224564 - 0.14414388
Average (Standard dev.)-0.000015270758 (±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

-
Supplemental data

-
Sample components

-
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

-
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

-
Supramolecule #2: ALG6

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

-
Supramolecule #3: 6AG9 Fab

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

-
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

-
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

-
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

-
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

-
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

-
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

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE-PROPANE

-
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

+
Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 171764
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more