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- EMDB-75118: Chloroplast Glutamyl Peptidase D855N in open-closed conformation -

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Open data


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Basic information

Entry
Database: EMDB / ID: EMD-75118
TitleChloroplast Glutamyl Peptidase D855N in open-closed conformation
Map data
Sample
  • Complex: Chloroplast glutamyl endopeptidase dimeric protein
    • Protein or peptide: Isoform 2 of Probable glutamyl endopeptidase, chloroplastic
KeywordsS9 protease / enzyme / serine protease / alpha-beta-alpha sandwich fold / beta-propeller / PLANT PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsEhrlich JJ / Routray P / van Wijk KJ / Kawate T
Funding support United States, 3 items
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, 2026-
Header (metadata) releaseMay 27, 2026-
Map releaseMay 27, 2026-
UpdateMay 27, 2026-
Current statusMay 27, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_75118.png

Downloads & links

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Map

FileDownload / File: emd_75118.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.37 Å/pix.
x 208 pix.
= 284.96 Å		1.37 Å/pix.
x 208 pix.
= 284.96 Å		1.37 Å/pix.
x 208 pix.
= 284.96 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.37 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.3874937 - 0.6662021
Average (Standard dev.)-0.00000831723 (±0.013577166)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions208208208
Spacing208208208
CellA=B=C: 284.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_75118_msk_1.map
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Additional map: Raw unmasked, unfiltered map

Fileemd_75118_additional_1.map
AnnotationRaw unmasked, unfiltered map
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Half map: #2

Fileemd_75118_half_map_1.map
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Half map: #1

Fileemd_75118_half_map_2.map
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Sample components

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Entire : Chloroplast glutamyl endopeptidase dimeric protein

EntireName: Chloroplast glutamyl endopeptidase dimeric protein
Components
  • Complex: Chloroplast glutamyl endopeptidase dimeric protein
    • Protein or peptide: Isoform 2 of Probable glutamyl endopeptidase, chloroplastic

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Supramolecule #1: Chloroplast glutamyl endopeptidase dimeric protein

SupramoleculeName: Chloroplast glutamyl endopeptidase dimeric protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 104.912 KDa

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

MacromoleculeName: Isoform 2 of Probable glutamyl endopeptidase, chloroplastic
type: protein_or_peptide / ID: 1
Details: Catalytically dead AtCGEP D855N recombinantly expressed in E. coli and purified with an N-terminal 6x HIS tag followed by a GG linker
Number of copies: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 100.357625 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: HHHHHHGGAS RSASRLRSLA SACSGGAEDG GGTSNGSLSA SATATEDDEL AIGTGYRLPP PEIRDIVDAP PVPALSFSPH RDKILFLKR RALPPLADLA RPEEKLAGVR IDGYCNTRSR MSFYTGLGIH QLLPDGTLSP EKEITGIPDG GKINFVTWSN D GKHLAFSI ...String:
HHHHHHGGAS RSASRLRSLA SACSGGAEDG GGTSNGSLSA SATATEDDEL AIGTGYRLPP PEIRDIVDAP PVPALSFSPH RDKILFLKR RALPPLADLA RPEEKLAGVR IDGYCNTRSR MSFYTGLGIH QLLPDGTLSP EKEITGIPDG GKINFVTWSN D GKHLAFSI RVDENGNSSK PVVWVADVET GVARPLFNSQ DIFLNAIFES FVWIDNSTLL VSTIPSSRGE PPKKPLVPSG PK TLSNETK TVVQVRTFQD LLKDEYDADL FDYYASSQLV LASLDGTVKE VGVPAVYTSL DPSTDHKYLL VSSLHRPYSF IVP CGRFPK KVEVWTTDGR FVRQLCDLPL AEDIPIASNS VRKGMRSINW RADKPSTLYW AETQDGGDAK MEVSPRDIVY MQSA EPLAG EEPEVLHKLD LRYGGISWCD DTLALVYESW YKTRRTRTWV ISPGSNDVSP RILFDRSSED VYSDPGSTML RRTDA GTYV IAKIKKENDE GTYVLLNGSG ATPQGNVPFL DLFDINTGNK ERIWESDKEK YFETVVALMS DQKEGDLKME ELKILT SKE SKTENTQYSL QLWPDRKVQQ ITNFPHPYPQ LASLQKEMIR YQRKDGVQLT ATLYLPPGYD PSKDGPLPCL FWSYPGE FK SKDAAGQVRG SPNEFAGIGS TSALLWLARR FAILSGPTIP IIGEGDEEAN DRYVEQLVAS AEAAVEEVVR RGVADRSK I AVGGHSYGAF MTANLLAHAP HLFACGIARS GAYNRTLTPF GFQNEDRTLW EATNVYVEMS PFMSANKIKK PILLIHGEE NNNPGTLTMQ SDRFFNALKG HGALCRLVVL PHESHGYSAR ESIMHVLWET DRWLQKYCVP NTSDADTSPD QSKEGSDSAD KVSTGTGGG NPEFGEHEVH SKLRRSLL

UniProtKB: Probable glutamyl endopeptidase, chloroplastic

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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

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H13Cl2NO3Tris-HCl
100.0 mMNaClsodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 10.1325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 7 force, 4 seconds, no wait time or drain time.
DetailsIMAC purified catalytic dead CGEP recombinantly expressed in e.coli

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON I (4k x 4k) / Number grids imaged: 1 / Number real images: 4206 / Average electron dose: 50.0 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: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 96000
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

Particle selectionNumber selected: 2090336
CTF correctionSoftware - Name: CTFFIND (ver. 4.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: Generated from Ab initio reconstruction of high resolution particle stack separated with 2D classification
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.0) / Software - details: Local refinement / Number images used: 70609
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.0) / Software - details: Ab initio
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.0) / Software - details: Local refinement

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