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- PDB-29te: The complex of A1AT-NHK with the ERAD misfolded glycoprotein chec... -

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Basic information

Entry
Database: PDB / ID: 29te
TitleThe complex of A1AT-NHK with the 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 / serpin / Alpha1 antitrypsin / null Hong Kong / A1AT
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
beta-D-mannopyranose / KIFUNENSINE / alpha-D-mannopyranose / 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: 3.46 Å
AuthorsRoversi, P. / Hitchman, C.J. / Gooptu, B. / Bhogadia, M. / Lia, A.
Funding support United Kingdom, Italy, 4items
OrganizationGrant numberCountry
Wellcome Trust204801/Z/16/Z United Kingdom
Wellcome Trust214090/Z/18/Z United Kingdom
Italian National Research Council (CNR)Plant_EDEM Italy
Italian National Research Council (CNR)B53C22009060005 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
DepositionApr 7, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 24, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 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

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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)203,99919
Polymers198,0992
Non-polymers5,89917
Water00
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: N-terminal GFP-fuson of the Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM),Green fluorescent protein N-terminally fused to alpha-1,2-Mannosidase
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: 55936.445 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_2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: G0SGS2, protein disulfide-isomerase

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Sugars , 6 types, 15 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.: 3
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 alpha-D-mannopyranose-(1-6)-alpha-D-mannopyranose


Type: oligosaccharide / Mass: 342.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-6DManpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a1122h-1a_1-5]/1-1/a6-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-Manp]{[(6+1)][a-D-Manp]{}}LINUCSPDB-CARE
#5: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#6: Sugar
ChemComp-MAN / alpha-D-mannopyranose / alpha-D-mannose / D-mannose / mannose


Type: D-saccharide, alpha linking / Mass: 180.156 Da / Num. of mol.: 5 / 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
#9: 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
#10: Sugar ChemComp-BMA / beta-D-mannopyranose / beta-D-mannose / D-mannose / mannose


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

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Non-polymers , 2 types, 2 molecules

#7: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-KIF / KIFUNENSINE


Mass: 232.191 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H12N2O6 / Feature type: SUBJECT OF INVESTIGATION / Comment: inhibitor, alkaloid*YM

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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
1Ternary complex of human A1AT-NH and Chaetomium thermophilum Endoplasmic reticulum degradation enhancing mannosidase (CtEDEM) plus Endoplasmic reticulum degradation enhancing protein disulfide isomerase (CtPDI)COMPLEXCoexpressed by transient co-transfection of three plasmids. A1AT is in the map but was not modelled#1-#20RECOMBINANT
2Green fluorescent protein N-terminally fused to alpha-1,2-MannosidaseCOMPLEX#11RECOMBINANT
3Protein disulfide isomeraseCOMPLEX#21RECOMBINANT
4Human Alpha1-antitrypsin Null Hong Kong mutant. Sequence: MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLRGHRGGTPEALQGRACOMPLEXThe protein is in the sample and appears as a low resolution volume in the map - but no atomic model for it was built.1RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.22 MDaYES
210.15 MDaNO
310.07 MDaNO
410.055 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDOrganelle
21Thermochaetoides thermophila (fungus)209285Endoplasmic reticulum
32Thermochaetoides thermophila (fungus)209285Endoplasmic reticulum
43Thermochaetoides thermophila (fungus)209285Endoplasmic reticulum
54Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Homo sapiens (human)9606HEK293FCtEDEM-pHLsec, CtPDI-pHLsec
32Homo sapiens (human)9606HEK293FpHLsec_CtEDEM
43Homo sapiens (human)9606HEK293FpHLsec_CtPDI
54Homo sapiens (human)9606HEK293F
Buffer solutionpH: 7.5 / Details: 20 mM HEPES pH 7.5, 100 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMSodium ChlorideNaCl1
220 mMSodium HEPESC8H17N2NaO4S1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 0.5 mL of sample was purified on a Superdex 200 Increase 10/300 GL (SEC, Cytiva - 28990944, USA) equilibrated against SEC buffer. Fractions containing the complex were identified from the ...Details: 0.5 mL of sample was purified on a Superdex 200 Increase 10/300 GL (SEC, Cytiva - 28990944, USA) equilibrated against SEC buffer. Fractions containing the complex were identified from the SEC chromatogram and SDS-PAGE and taken forward for Cryo-EM. Concentration of each fraction was measured using the A280 nm reading.
Specimen supportDetails: 50 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: PROPANE / Humidity: 100 % / Chamber temperature: 277 K
Details: 3 uL of protein were added to the grid for 1 minute, at 4 degrees C with 100% humidity, before blotting with Whatman No1 filter paper (Sigma-aldrich, Germany) and washing three times with 20 ...Details: 3 uL of protein were added to the grid for 1 minute, at 4 degrees C with 100% humidity, before blotting with Whatman No1 filter paper (Sigma-aldrich, Germany) and washing three times with 20 uL drops of ultrapure water. Blot force 10 for 3 s before plunge freezing immediately into liquid ethane. A 30 second wait time after sample application before plunge freezing.

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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 / Calibrated 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 recordingAverage exposure time: 3 sec. / Electron dose: 11.15 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2PHENIX1.21.2_5419model refinement
3EPUimage acquisition
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 123770
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 765 / Algorithm: BACK PROJECTION / Num. of class averages: 4 / Symmetry type: POINT
RefinementHighest resolution: 3.46 Å / Cross valid method: NONE
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212548
ELECTRON MICROSCOPYf_angle_d0.53917088
ELECTRON MICROSCOPYf_dihedral_angle_d5.3542040
ELECTRON MICROSCOPYf_chiral_restr0.041970
ELECTRON MICROSCOPYf_plane_restr0.0042191

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