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- PDB-9xfh: Cryo-EM structure of Ceg14 and Actin complex -

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Basic information

Entry
Database: PDB / ID: 9xfh
TitleCryo-EM structure of Ceg14 and Actin complex
Components
  • Actin
  • Ceg14
KeywordsCELL INVASION / Legionella / Ceg14 / Actin
Function / homologyADENOSINE-5'-TRIPHOSPHATE
Function and homology information
Biological speciesLegionella (bacteria)
Lepus (hares)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsLi, Y. / Zheng, Q. / Li, S.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2026
Title: Molecular basis of host ATP level modulation by actin-dependent secreted bacterial ATPase and its metaeffector.
Authors: Hongxin Guan / Yu Li / Luhao Zhang / Song Xie / Yuchen Jiang / Chunlin He / Fan Li / Yin-di Jiao / Mengrou Gan / Yiran Sha / Fengjie Zheng / Kaiqiong Zhang / Zhao-Qing Luo / Qi Lai / Jinfu ...Authors: Hongxin Guan / Yu Li / Luhao Zhang / Song Xie / Yuchen Jiang / Chunlin He / Fan Li / Yin-di Jiao / Mengrou Gan / Yiran Sha / Fengjie Zheng / Kaiqiong Zhang / Zhao-Qing Luo / Qi Lai / Jinfu Su / Ningshao Xia / Qingbing Zheng / Jinyu Li / Shaowei Li / Songying Ouyang /
Abstract: Legionella pneumophila employs effectors including kinases, phosphoryl-AMPylases, ATPases, etc. to exploit host ATP for infection. Ceg14, a member of the S-HxxxE family, modulates host-cell energy ...Legionella pneumophila employs effectors including kinases, phosphoryl-AMPylases, ATPases, etc. to exploit host ATP for infection. Ceg14, a member of the S-HxxxE family, modulates host-cell energy levels in concert with host actin and the metaeffector AnkJ: actin activates while AnkJ inhibits Ceg14 ATPase activity. However, the molecular basis of this regulation remains unclear. Here we present Cryo-EM structures of Ceg14-actin, Ceg14-AnkJ, and Ceg14-actin-AnkJ complexes at 2.89 Å, 2.93 Å, and 2.52 Å, respectively. Actin binds to the C-terminal α-helix of the Ceg14 catalytic domain (CD), inducing rearrangement of its N-terminal domain (NTD) and reconfiguration of the flexible Lid domain. Surprisingly, AnkJ binds to the surface opposite the catalytic pocket rather than occupying the pocket. Using integrated in silico, in vitro, and in cellulo approaches, we propose a mechanism for Ceg14-mediated ATP hydrolysis. Actin binding triggers NTD rotation, driving the catalytic pocket through open, intermediate, and closed states. In the open state, H571 catalyzes ATP conversion to AMP and PPi. AnkJ binding to an allosteric site locks the intermediate state, thereby inhibiting hydrolysis. Together, our study reveals the molecular mechanism by which actin and AnkJ reciprocally regulate Ceg14 activity.
History
DepositionOct 28, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 9, 2026Provider: repository / Type: Initial release
Revision 1.0Sep 9, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ceg14
C: Actin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)118,5873
Polymers118,0802
Non-polymers5071
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 Ceg14


Mass: 75982.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Legionella (bacteria) / Production host: Escherichia coli (E. coli)
#2: Protein Actin


Mass: 42096.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lepus (hares) / Production host: Lepus (hares)
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestY
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: Ceg14 and Actin complex / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Source (natural)Organism: Legionella (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 236934 / Symmetry type: POINT
RefinementStereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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