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- EMDB-17749: The ERAD misfolded glycoprotein checkpoint complex from Chaetomiu... -

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Basic information

Entry
Database: EMDB / ID: EMD-17749
TitleThe ERAD misfolded glycoprotein checkpoint complex from Chaetomium thermophilum (EDEM:PDI heterodimer).
Map dataCryosparc class, 150,000 particles, iteration 4
Sample
  • Complex: Complex of Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM) and Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)
    • Complex: Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM)
      • Protein or peptide: Green fluorescent protein,alpha-1,2-Mannosidase
    • Complex: Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)
      • Protein or peptide: Protein disulfide-isomerase
  • Ligand: CALCIUM ION
  • Ligand: THIOSULFATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: alpha-D-mannopyranose
  • Ligand: water
Keywordsmannosidase / disulfide isomerase / glycoprotein degradation / erad / misfolding / OXIDOREDUCTASE
Function / homology
Function and homology information


endoplasmic reticulum mannose trimming / mannosyl-oligosaccharide 1,2-alpha-mannosidase activity / protein disulfide-isomerase / endoplasmic reticulum quality control compartment / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / protein disulfide isomerase activity / protein glycosylation / ERAD pathway / bioluminescence / generation of precursor metabolites and energy ...endoplasmic reticulum mannose trimming / mannosyl-oligosaccharide 1,2-alpha-mannosidase activity / protein disulfide-isomerase / endoplasmic reticulum quality control compartment / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / protein disulfide isomerase activity / protein glycosylation / ERAD pathway / bioluminescence / generation of precursor metabolites and energy / carbohydrate metabolic process / endoplasmic reticulum lumen / calcium ion binding / 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-1,2-Mannosidase / Protein disulfide-isomerase / Green fluorescent protein
Similarity search - Component
Biological speciesThermochaetoides thermophila (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsRoversi P / Hitchman CJ / Lia A / Bayo Y
Funding support United Kingdom, Italy, 3 items
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 CJ / Lia A / Bayo Y
History
DepositionJun 27, 2023-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17749.png

Downloads & links

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Map

FileDownload / File: emd_17749.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryosparc class, 150,000 particles, iteration 4
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 300 pix.
= 250.5 Å		0.84 Å/pix.
x 300 pix.
= 250.5 Å		0.84 Å/pix.
x 300 pix.
= 250.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.30968925 - 0.64553756
Average (Standard dev.)0.0002195782 (±0.017497346)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 250.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryosparc class, 150,000 particles, iteration 3

Fileemd_17749_half_map_1.map
AnnotationCryosparc class, 150,000 particles, iteration 3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryosparc class, 150,000 particles, iteration 3

Fileemd_17749_half_map_2.map
AnnotationCryosparc class, 150,000 particles, iteration 3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of Chaetomium thermophilum Endoplasmic reticulum degradat...

EntireName: Complex of Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM) and Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)
Components
  • Complex: Complex of Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM) and Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)
    • Complex: Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM)
      • Protein or peptide: Green fluorescent protein,alpha-1,2-Mannosidase
    • Complex: Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)
      • Protein or peptide: Protein disulfide-isomerase
  • Ligand: CALCIUM ION
  • Ligand: THIOSULFATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: alpha-D-mannopyranose
  • Ligand: water

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Supramolecule #1: Complex of Chaetomium thermophilum Endoplasmic reticulum degradat...

SupramoleculeName: Complex of Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM) and Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Coexpressed by transient co-transfection of two plasmids
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Molecular weightTheoretical: 55 KDa

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Supramolecule #2: Chaetomium thermophilum Endoplasmic reticulum degradation enhanci...

SupramoleculeName: Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM)
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: CtEDEM expressed as a N-term GFP fusion, truncated at the N-term and with hexahistidine tag at the Cterm
Source (natural)Organism: Thermochaetoides thermophila (fungus) / Location in cell: ER

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Supramolecule #3: Endoplasmic reticulum degradation enhancing protein disulfide iso...

SupramoleculeName: Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: CtPDI expressed as a Cterm truncation with no KDEL and a hexahistidine tag at the Cterm.
Source (natural)Organism: Thermochaetoides thermophila (fungus)

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

MacromoleculeName: Green fluorescent protein,alpha-1,2-Mannosidase / type: protein_or_peptide / ID: 1
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)
Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Molecular weightTheoretical: 142.162812 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MVSKGELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNYNSHN VYIMADKQKN G IKVNFKIR ...String:
MVSKGELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNYNSHN VYIMADKQKN G IKVNFKIR HNIEDGSVQL ADHYQQNTPI GDGPVLLPDN HYLSTQSALS KDPNEKRDHM VLLEFVTAAG ITLGMDELYK LE VLFQGPD RVRALRRETV EMFYYGFDNY MKVAFPEDEL RPVSCTPLTR DLKNPRNFEL NDVLGNYSLT LIDSLSTLAI LAS APAEDS GTGPKALRDF QDGVAALVEQ YGDGRPGPSG VGRRARGFDL DSKVQVFETV IRGVGGLLSA HLFAIGALPI TGYQ PLRQE DDLFNPPPIP WPNGFTYDGQ LLRLALDLAQ RLLPAFYTKT GLPYPRVNLR HGIPFYVNSP LHEDPPAKGT TEGPP EITE TCSAGAGSLV LEFTVLSRLT GDPRFEQAAK RAFWAVWYRK SQIGLIGAGV DAEQGHWIGT YSVIGAGADS FFEYAL KSH ILLSGHALPN QTHPSPLHKD VNWMDPNTLF EPLSDAENSA ESFLEAWHHA HAAIKRHLYS EREHPHYDNV NLWTGSL VS HWVDSLGAYY SGLLVLAGEV DEAIETNLLY AAIWTRYAAL PERWSLREKT VEGGLGWWPL RPEFIESTYH LYRATKDP W YLYVGEMVLR DITRRCWTPC GWAGLQNVLS GEKSDRMESF FLGETTKYMY LLFDDDHPLN KLDASFVFTT EGHPLILPK PKSARRSRNS PRSSQKALTV YQGEGFTNSC PPRPSITPLS GSVIAARDDI YHPARMVDLH LLTTSKHALD GGQMSGQHMA KSNYTLYPW TLPPELLPSN GTCAKVYQPH EVTLEFASNT QQVLGGSAFN FMLSGQNLER LSTDRIRVLS LSGLKITLQL V EEGEREWR VTKLNGIPLG RDEYVVINRA ILGDVSDPRF NLVRDPVIAK LQQLHQVNLL DDTTTEEHPD NLDTLDTASA ID LPQDQSS DSEVPDPANL SALLPDLSSF VKSLFARLSN LTSPSPDPSS NLPLNVVINQ TAILPTGIGA APLPPAASNS PSG APIPVF GPVPESLFPW KTIYAAGEAC AGPLPDSAPR ENQVILIRRG GCSFSDKLAN IPAFTPSEES LQLVVVVSDD EHEG QSGLV RPLLDEIQHT PGGMPRRHPI AMVMVGGGET VYQQLSVASA IGIQRRYYIE SSGVKVKNII VDDGDGGVDG GTKHH HHHH

UniProtKB: Green fluorescent protein, alpha-1,2-Mannosidase

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Macromolecule #2: Protein disulfide-isomerase

MacromoleculeName: Protein disulfide-isomerase / type: protein_or_peptide / ID: 2
Details: Chaetomium thermophilum Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)
Number of copies: 1 / Enantiomer: LEVO / EC number: protein disulfide-isomerase
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Molecular weightTheoretical: 55.841367 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ETGSDVIQLK KDTFDDFIKS NDLVLAEFFA PWCGHCKALA PEYEEAATNL KDKNIKLVKV DCTEETELCQ EHGVEGYPTL KVFRGLDNV TPYKGQRKAA AITSYMIKQS LPAVSDVTKD TLEEFKKADK VVLVAYVDAS DKASAEVFKK VAEKLRDNYP F GSSSDAEL ...String:
ETGSDVIQLK KDTFDDFIKS NDLVLAEFFA PWCGHCKALA PEYEEAATNL KDKNIKLVKV DCTEETELCQ EHGVEGYPTL KVFRGLDNV TPYKGQRKAA AITSYMIKQS LPAVSDVTKD TLEEFKKADK VVLVAYVDAS DKASAEVFKK VAEKLRDNYP F GSSSDAEL AEAEGVKAPA IVLYKDFDEG KAVFTEKFDE EAIQKWAKVA ATPLIGEIGP ETYGEYMAAG IPLAYIFAET PE ERKELSE KLKPIAEATR GKINFGTIDA KAYGAHAGNL NLKTDKFPAF AIQETTKNQK FPYDQDKEIT HDSIKQFVDD YLA GKIEPS IKSEPIPEKQ EGPVTVVVAK TYNDIVLDDT KDVLIEFYAP WCGHCKALAP KYEELGRLYS NSEFKDRVVI AKID ATAND VPDDIMGFPT IKMYPAGAKD KPVTYSGNRS VEDMIKFVAE NGKYKALISE NEEENATAAS SSSETSATPT STAAS EETA ASETASAEEG KETAGTKHHH HHH

UniProtKB: Protein disulfide-isomerase

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #6: THIOSULFATE

MacromoleculeName: THIOSULFATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: THJ
Molecular weightTheoretical: 112.128 Da
Chemical component information

ChemComp-THJ:
THIOSULFATE

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Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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Macromolecule #8: alpha-D-mannopyranose

MacromoleculeName: alpha-D-mannopyranose / type: ligand / ID: 8 / Number of copies: 1 / Formula: MAN
Molecular weightTheoretical: 180.156 Da
Chemical component information

ChemComp-MAN:
alpha-D-mannopyranose

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 3 / 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

Concentration0.02 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMC6H13NO4SMES
1.0 mMCaCl2calcium chloride

Details: Micro SEC buffer: 150 mM NaCl, 20 mM MES pH 7.0, 1 mM CaCl2
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.2 kPa
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 ...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.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Blotting time 3 s, waiting time 30 s Sample volume 3 uL, blot force 10.
Details40 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 1400000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Avg.num./class: 150000 / Software - Name: cryoSPARC

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