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- PDB-10eu: Chloroplast Glutamyl Peptidase D855N in open-open conformation -

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Basic information

Entry
Database: PDB / ID: 10eu
TitleChloroplast Glutamyl Peptidase D855N in open-open conformation
ComponentsIsoform 2 of Probable glutamyl endopeptidase, chloroplastic
KeywordsPLANT PROTEIN / S9 protease / enzyme / serine protease / alpha-beta-alpha sandwich fold / beta-propeller
Function / homology
Function and homology information


chloroplast stroma / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / chloroplast / serine-type endopeptidase activity / proteolysis / cytosol
Similarity search - Function
Peptidase S9, prolyl oligopeptidase, catalytic domain / Prolyl oligopeptidase family / Six-bladed beta-propeller, TolB-like / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Probable glutamyl endopeptidase, chloroplastic
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsEhrlich, J.J. / Routray, P. / van Wijk, K.J. / Kawate, T.
Funding support United States, 3items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)2222495 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10OD030470 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R24GM154185 United States
CitationJournal: Protein Sci / Year: 2026
Title: Structural basis for dimerization, catalytic regulation, and substrate selectivity of the chloroplast S9D CGEP protease in Arabidopsis thaliana.
Authors: Jacqueline J Ehrlich / Pratyush Routray / Louis Enns / Klaas J van Wijk / Toshimitsu Kawate /
Abstract: S9 proteases are widely distributed across the tree-of-life and play essential roles in protein processing. However, the structural and mechanistic basis for protease activity in the S9D subfamily, ...S9 proteases are widely distributed across the tree-of-life and play essential roles in protein processing. However, the structural and mechanistic basis for protease activity in the S9D subfamily, restricted to photosynthetic eukaryotes (e.g., plants), cyanobacteria, proteobacteria and flavobacteria, is unknown. Here, we report the first high-resolution cryo-EM structures of an S9D protease, chloroplast glutamyl endopeptidase (CGEP) from the model plant Arabidopsis thaliana. CGEP adopts a dimeric architecture stabilized by two distinct interfaces: hydrophobic interactions between catalytic domains and an interdomain β-sheet linking the cap and catalytic domains. These interactions create a scaffold that supports a hinge loop, which acts as a steric gate to restrict substrate access and confine catalytic activity to the closed conformation. Unlike S9A-B-C proteases, CGEP maintains an intact catalytic triad in both open and closed states, relying on hinge-loop gating rather than catalytic disruption for regulation. Structural analysis and mutagenesis reveal that the hinge loop forms a conserved pocket favoring glutamate side chains, explaining CGEP's strong glutamate preference at cleavage sites. Together, these findings uncover a unique regulatory paradigm for S9D proteases and provide a structural framework for understanding substrate selectivity and dimerization.
History
DepositionJan 15, 2026Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 27, 2026Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 27, 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: Isoform 2 of Probable glutamyl endopeptidase, chloroplastic
B: Isoform 2 of Probable glutamyl endopeptidase, chloroplastic


Theoretical massNumber of molelcules
Total (without water)200,7152
Polymers200,7152
Non-polymers00
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

#1: Protein Isoform 2 of Probable glutamyl endopeptidase, chloroplastic


Mass: 100357.625 Da / Num. of mol.: 2 / Mutation: D855N
Source method: isolated from a genetically manipulated source
Details: Catalytically dead AtCGEP D855N recombinantly expressed in E. coli and purified with an N-terminal 6x HIS tag followed by a GG linker
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: GEP, At2g47390, T8I13.23 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: Q8VZF3, Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
Has protein modificationN

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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

ComponentName: Chloroplast glutamyl endopeptidase dimeric protein / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.104912 MDa / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HClC4H13Cl2NO31
2100 mMsodium chlorideNaCl1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: IMAC purified catalytic dead CGEP recombinantly expressed in e.coli
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 7 force, 4 seconds, no wait time or drain time

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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: 96000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON I (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4206

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.7.0particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7Coot0.9.8.5model fitting
9PHENIX1.21.2_5419model refinement
10cryoSPARC4.7.0initial Euler assignmentAb initio
11cryoSPARC4.7.0final Euler assignmentLocal refinement
13cryoSPARC4.7.03D reconstructionLocal refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2090336
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55962 / Symmetry type: POINT
RefinementHighest resolution: 3 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212910
ELECTRON MICROSCOPYf_angle_d0.5117562
ELECTRON MICROSCOPYf_dihedral_angle_d2.9951744
ELECTRON MICROSCOPYf_chiral_restr0.041916
ELECTRON MICROSCOPYf_plane_restr0.0042288

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