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- PDB-9tpa: The ERAD misfolded glycoprotein checkpoint complex from Chaetomiu... -

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Basic information

Entry
Database: PDB / ID: 9tpa
TitleThe ERAD misfolded glycoprotein checkpoint complex from Chaetomium thermophilum (EDEM:PDI heterodimer).
Components
  • Green fluorescent protein,alpha-1,2-Mannosidase
  • Protein disulfide-isomerase
KeywordsHYDROLASE / mannosidase / disulfide isomerase / glycoprotein degradation / erad / misfolding / OXIDOREDUCTASE
Function / homology
Function and homology information


mannosyl-oligosaccharide 1,2-alpha-mannosidase activity / endoplasmic reticulum mannose trimming / 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-disulfide reductase activity / ERAD pathway / response to endoplasmic reticulum stress / bioluminescence ...mannosyl-oligosaccharide 1,2-alpha-mannosidase activity / endoplasmic reticulum mannose trimming / 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-disulfide reductase activity / ERAD pathway / response to endoplasmic reticulum stress / bioluminescence / generation of precursor metabolites and energy / : / protein folding / 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 family active 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 family active site. / Thioredoxin, conserved site / Thioredoxin domain profile. / Thioredoxin domain / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / 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 speciesAequorea victoria (jellyfish)
Thermochaetoides thermophila (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsRoversi, P. / Hitchman, C.J. / Lia, A. / Bayo, Y. / Gooptu, B.
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: Biorxiv / Year: 2025
Title: Structure and function of the EDEM:PDI ERAD checkpoint complex
Authors: Hitchman, C.J. / Lia, A. / Chiritoiu, G.N. / Munteanu, C.V.A. / Ortigosa, J.R. / Ghenea, S. / Savva, C. / Wada, I. / De Benedictis, M. / Tax, G. / Bayo, Y. / Crescioli, I. / Alonzi, D.L. / ...Authors: Hitchman, C.J. / Lia, A. / Chiritoiu, G.N. / Munteanu, C.V.A. / Ortigosa, J.R. / Ghenea, S. / Savva, C. / Wada, I. / De Benedictis, M. / Tax, G. / Bayo, Y. / Crescioli, I. / Alonzi, D.L. / Quigley, A. / Modenutti, C.P. / Petrescu, S.M. / Santino, A. / Gooptu, B. / Hosokawa, N. / Roversi, P.
History
DepositionDec 17, 2025Deposition site: PDBE / Processing site: PDBE
SupersessionJul 1, 2026ID: 8PKO
Revision 1.0Jul 1, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Primary map / Data content type: Primary map / Provider: 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)201,06112
Polymers198,0042
Non-polymers3,05710
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
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)
Source: (gene. exp.) Aequorea victoria (jellyfish), (gene. exp.) Thermochaetoides thermophila (fungus)
Gene: GFP, CTHT_0058730 / Plasmid: pHLsec_GFP-CtEDEM / 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 C-terminal ER retention signal
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: pHLsec_CtEDEM / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: G0SGS2, protein disulfide-isomerase

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Sugars , 4 types, 8 molecules

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


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+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 / Feature type: SUBJECT OF INVESTIGATION
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.: 3 / Source method: obtained synthetically / Formula: C6H12O6 / Feature type: SUBJECT OF INVESTIGATION
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: obtained synthetically / 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
Has protein modificationY

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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
IDNameTypeDetails (eV)Entity 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
11Thermochaetoides thermophila (fungus)209285
22Thermochaetoides thermophila (fungus)209285ER
33Thermochaetoides thermophila (fungus)209285
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
11Homo sapiens (human)9606HEK293FCtEDEM-pHLsec and CtPDI-pHLsec
22Homo 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 supportGrid 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: TFS 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
1Topazparticle selection
2EPUimage acquisition
8PHENIXmodel refinement
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
Atomic model buildingSource name: AlphaFold / Type: other
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 126.76 Å2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms11233 0 195 3 11431
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002411710
ELECTRON MICROSCOPYf_angle_d0.510215936
ELECTRON MICROSCOPYf_chiral_restr0.04191789
ELECTRON MICROSCOPYf_plane_restr0.00472061
ELECTRON MICROSCOPYf_dihedral_angle_d6.89991762

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