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- EMDB-66735: Cryo-EM structure of Ceg14-AnkJ-Actin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-66735
TitleCryo-EM structure of Ceg14-AnkJ-Actin complex
Map data
Sample
  • Complex: Ceg14-AnkJ-Actin complex
    • Protein or peptide: Actin
    • Protein or peptide: Ceg14
    • Protein or peptide: AnkJ
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsLegionella / Ceg14 / AnkJ / Actin / CELL INVASION
Biological speciesLegionella (bacteria) / Lepus (hares)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.52 Å
AuthorsLi Y / Li S / Zheng Q / Wu Y
Funding support1 items
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 26, 2025-
Header (metadata) releaseSep 9, 2026-
Map releaseSep 9, 2026-
UpdateSep 9, 2026-
Current statusSep 9, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_66735.png

Downloads & links

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Map

FileDownload / File: emd_66735.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 324 pix.
= 210.6 Å		0.65 Å/pix.
x 324 pix.
= 210.6 Å		0.65 Å/pix.
x 324 pix.
= 210.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.0017601077 - 2.1664534
Average (Standard dev.)0.00223514 (±0.035054002)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 210.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_66735_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #2

Fileemd_66735_half_map_2.map
Projections & Slices
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Sample components

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Entire : Ceg14-AnkJ-Actin complex

EntireName: Ceg14-AnkJ-Actin complex
Components
  • Complex: Ceg14-AnkJ-Actin complex
    • Protein or peptide: Actin
    • Protein or peptide: Ceg14
    • Protein or peptide: AnkJ
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Ceg14-AnkJ-Actin complex

SupramoleculeName: Ceg14-AnkJ-Actin complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Legionella (bacteria)

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Macromolecule #1: Actin

MacromoleculeName: Actin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lepus (hares)
Molecular weightTheoretical: 42.096953 KDa
Recombinant expressionOrganism: Lepus (hares)
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSSSL EK SYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQK EIT ALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

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Macromolecule #2: Ceg14

MacromoleculeName: Ceg14 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Legionella (bacteria)
Molecular weightTheoretical: 75.982969 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQNLDEILKK LRKDLKKTSK ETTEKLSVLS KKVAEDVVKV SNQTVQKINE VTKKFTVDTT VSTIDTQKIL DDFMKNIRFS RFKQIWQAI EKTPKSKHIL LKSLFLKGLL THNKPLLEEI IRQIELIPYS SDLEMLGAFS QDKAHPLSPE LLEFVQEMLA N ESEKVLLT ...String:
MQNLDEILKK LRKDLKKTSK ETTEKLSVLS KKVAEDVVKV SNQTVQKINE VTKKFTVDTT VSTIDTQKIL DDFMKNIRFS RFKQIWQAI EKTPKSKHIL LKSLFLKGLL THNKPLLEEI IRQIELIPYS SDLEMLGAFS QDKAHPLSPE LLEFVQEMLA N ESEKVLLT ILMNAFQSIP ELSLDSKTGT LSMKTKKALL PLLIEKVDTS IAKSIMSQLT DISFDSVLPA FRPSIGDPSY QK TNINLNK YLSLVGNKTD ISEFLILTIG LFTSLKIGDK GFLQELSADY LFVRYDDCLH KIIEKLKEKE VIEQLGEKEV QKI LEASGN LKRFKPTSNN PRRFFETRLA QYINGLSHSE KTIEIDSIGE DKEPEDEELR DNTLKACNKI LQFFLGDCGR VDTP REMEQ KICIFANGYK DTSKFTKKEV EFITGVLHHA GAHKGRDRVE RDKLTGIKER KFETDSEKVG SDVWNCGGGV MKGCS PVFY DAQRDPMRNM LVDSSTVAPD NKEGKQWFWN NNRQYSSYVG SISGHTCNIV GMLAKYMTEY KEDLDLQNDI NLFLIQ VIG VYAKRGFHAM LEVIDVLHDP YVQDIFKGYG VQVNLYSYFK ENPELAGFLQ HAMNDATTYT QALVNKKHVK EALKSNS FF SKKDEESGDK KNDHTPKDGT VYGC

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Macromolecule #3: AnkJ

MacromoleculeName: AnkJ / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Legionella (bacteria)
Molecular weightTheoretical: 30.693082 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIKMGRSEMK IASAELRELM KAVSEGHYET VNTILDKDPE LVNQYAPPTY DSPLARVLNK KHIDYKMLDI LVKHHVDFDY PINYHKETP IELACKNQDL QLFKYLVQHN APISEQAPHF LLVNSTNIKY LTEDKIKNTC EIIKLMGGLE AVSSKCDAEG N RFGEQARK ...String:
MIKMGRSEMK IASAELRELM KAVSEGHYET VNTILDKDPE LVNQYAPPTY DSPLARVLNK KHIDYKMLDI LVKHHVDFDY PINYHKETP IELACKNQDL QLFKYLVQHN APISEQAPHF LLVNSTNIKY LTEDKIKNTC EIIKLMGGLE AVSSKCDAEG N RFGEQARK SQLINRFGGI VKYDYMQLLQ SVYPIVDREV DAPTIHGSTE VLTNLLNKIR GQFSSKETYD QQNLKDSISL FF MTGGEIP PSRKVPESRF EEAGIDTPKN AL

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 280526
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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