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- PDB-8pko: The ERAD misfolded glycoprotein checkpoint complex from Chaetomiu... -

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Basic information

Entry
Database: PDB / ID: 8pko
TitleThe ERAD misfolded glycoprotein checkpoint complex from Chaetomium thermophilum (EDEM:PDI heterodimer).
Components
  • Green fluorescent protein,alpha-1,2-Mannosidase
  • Protein disulfide-isomerase
KeywordsOXIDOREDUCTASE / mannosidase / disulfide isomerase / glycoprotein degradation / erad / misfolding
Function / homology
Function and homology information


mannosyl-oligosaccharide 1,2-alpha-mannosidase activity / protein disulfide-isomerase / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / protein disulfide isomerase activity / protein glycosylation / response to endoplasmic reticulum stress / bioluminescence / generation of precursor metabolites and energy / protein folding / carbohydrate metabolic process ...mannosyl-oligosaccharide 1,2-alpha-mannosidase activity / protein disulfide-isomerase / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / protein disulfide isomerase activity / protein glycosylation / response to endoplasmic reticulum stress / bioluminescence / generation of precursor metabolites and energy / protein folding / carbohydrate metabolic process / endoplasmic reticulum lumen / calcium ion binding / cell surface / endoplasmic reticulum / membrane
Similarity search - Function
ER degradation-enhancing alpha-mannosidase-like protein 1/2/3 / Glycoside hydrolase family 47 / Seven-hairpin glycosidases / Glycosyl hydrolase family 47 / Protein disulphide isomerase / Thioredoxin-like domain / Disulphide isomerase / Thioredoxin / Six-hairpin glycosidase-like superfamily / Thioredoxin, conserved site ...ER degradation-enhancing alpha-mannosidase-like protein 1/2/3 / Glycoside hydrolase family 47 / Seven-hairpin glycosidases / Glycosyl hydrolase family 47 / Protein disulphide isomerase / Thioredoxin-like domain / Disulphide isomerase / Thioredoxin / Six-hairpin glycosidase-like superfamily / Thioredoxin, conserved site / Thioredoxin family active site. / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Thioredoxin domain profile. / Thioredoxin domain / Thioredoxin-like superfamily
Similarity search - Domain/homology
alpha-D-mannopyranose / THIOSULFATE / alpha-1,2-Mannosidase / Protein disulfide-isomerase / Green fluorescent protein
Similarity search - Component
Biological speciesThermochaetoides thermophila (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsRoversi, P. / Hitchman, C.J. / Lia, A. / Bayo, Y.
Funding support United Kingdom, Italy, 3items
OrganizationGrant numberCountry
Wellcome Trust204801/Z/16/Z United Kingdom
Wellcome Trust214090/Z/18/Z United Kingdom
Italian National Research Council (CNR)Plant_EDEM Italy
CitationJournal: To Be Published
Title: The ERAD misfolded glycoprotein checkpoint complex from Chaetomium thermophilum (EDEM:PDI heterodimer).
Authors: Roversi, P. / Hitchman, C.J. / Lia, A. / Bayo, Y.
History
DepositionJun 27, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Green fluorescent protein,alpha-1,2-Mannosidase
B: Protein disulfide-isomerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,02510
Polymers198,0042
Non-polymers3,0218
Water543
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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Protein , 2 types, 2 molecules AB

#1: Protein Green fluorescent protein,alpha-1,2-Mannosidase


Mass: 142162.812 Da / Num. of mol.: 1 / Mutation: N-term truncation and GFP fusion
Source method: isolated from a genetically manipulated source
Details: Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM),Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM),Chaetomium ...Details: Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM),Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM),Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM),Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM),Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM),Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM),Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM),Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM)
Source: (gene. exp.) Thermochaetoides thermophila (fungus) / Gene: GFP, CTHT_0058730 / Plasmid: CtEDEM_pHLsec / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
References: UniProt: P42212, UniProt: G0SCX7, Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds
#2: Protein Protein disulfide-isomerase


Mass: 55841.367 Da / Num. of mol.: 1 / Mutation: Deletion of Cterminal HDEL
Source method: isolated from a genetically manipulated source
Details: Chaetomium thermophilum Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)
Source: (gene. exp.) Thermochaetoides thermophila (fungus) / Gene: CTHT_0067360 / Plasmid: CtEDEM_pHLsec / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: G0SGS2, protein disulfide-isomerase

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Sugars , 4 types, 6 molecules

#3: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 2 / Source method: isolated from a natural source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#4: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#7: 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.: 2 / 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
#8: Sugar ChemComp-MAN / alpha-D-mannopyranose / alpha-D-mannose / D-mannose / mannose


Type: D-saccharide, alpha linking / Mass: 180.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H12O6
IdentifierTypeProgram
DManpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-D-mannopyranoseCOMMON NAMEGMML 1.0
a-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 3 types, 5 molecules

#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-THJ / THIOSULFATE


Mass: 112.128 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: O3S2
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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
IDNameTypeDetailsEntity IDParent-IDSource
1Complex of Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM) and Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)COMPLEXCoexpressed by transient co-transfection of two plasmids#1-#20RECOMBINANT
2Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM)COMPLEXCtEDEM expressed as a N-term GFP fusion, truncated at the N-term and with hexahistidine tag at the Cterm#11RECOMBINANT
3Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)COMPLEXCtPDI expressed as a Cterm truncation with no KDEL and a hexahistidine tag at the Cterm.#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.22 MDaYES
210.170 MDaYES
310.055 MDaYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular location
21Thermochaetoides thermophila (fungus)209285
32Thermochaetoides thermophila (fungus)209285ER
33Thermochaetoides thermophila (fungus)209285
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
21Homo sapiens (human)9606HEK293FCtEDEM-pHLsec and CtPDI-pHLsec
32Homo sapiens (human)9606HEK293FCtEDEM_pHLsec
33Homo sapiens (human)9606HEK293FCtEDEM-pHLsec and CtPDI-pHLsec
Buffer solutionpH: 7
Details: Micro SEC buffer: 150 mM NaCl, 20 mM MES pH 7.0, 1 mM CaCl2
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMMESC6H13NO4S1
31 mMcalcium chlorideCaCl21
SpecimenConc.: 0.02 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 40 uL of a protein sample with an OD_280 of 0.88 were injected onto a Cytiva Superdex 200 Increase 3.2/300 size-exclusion chromatography column, equilibrated in 150 mM NaCl, 20 mM MES pH 7. ...Details: 40 uL of a protein sample with an OD_280 of 0.88 were injected onto a Cytiva Superdex 200 Increase 3.2/300 size-exclusion chromatography column, equilibrated in 150 mM NaCl, 20 mM MES pH 7.0, 1 mM CaCl2, collecting 50 uL fractions.
Specimen supportDetails: The Quantifoil Au300 R1.2/1.3 Holey Carbon grids were glow discharged using the EMS GloQube. For grids to be coated in GO, the Graphene Oxide setting was used (0.2m Bar, 40 mA for 180 ...Details: The Quantifoil Au300 R1.2/1.3 Holey Carbon grids were glow discharged using the EMS GloQube. For grids to be coated in GO, the Graphene Oxide setting was used (0.2m Bar, 40 mA for 180 seconds). For GO coating, graphene oxide solution (Sigma) at 0.2 mg/ml was spun at 300 g for 30 s to remove large aggregates. 3 uL was added to the grids for 1 minute, before blotting with Whatman No1 filter paper and washing three times with 20 uL drops of ultrapure water.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Blotting time 3 s, waiting time 30 s Sample volume 3 uL, blot force 10

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1400000 / Algorithm: BACK PROJECTION / Num. of class averages: 4 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01611709
ELECTRON MICROSCOPYf_angle_d0.95515933
ELECTRON MICROSCOPYf_dihedral_angle_d6.3571555
ELECTRON MICROSCOPYf_chiral_restr0.0611791
ELECTRON MICROSCOPYf_plane_restr0.0072060

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