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- EMDB-47690: Cryo-EM structure of NOT1:NOT8:PieF -

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Basic information

Entry
Database: EMDB / ID: EMD-47690
TitleCryo-EM structure of NOT1:NOT8:PieF
Map datamap used for model refinement
Sample
  • Complex: NOT1:NOT8:PieF
    • Protein or peptide: Ubiquitin-like protein SMT3,CCR4-NOT transcription complex subunit 8
    • Protein or peptide: Dot/Icm T4SS effector PieF
    • Protein or peptide: CCR4-NOT transcription complex subunit 1
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsPieF / mRNA decay / deadenylation / CCR4-NOT / host-pathogen interactions / GENE REGULATION
Function / homology
Function and homology information


positive regulation of cytoplasmic mRNA processing body assembly / poly(A)-specific ribonuclease / poly(A)-specific ribonuclease activity / CCR4-NOT core complex / armadillo repeat domain binding / CCR4-NOT complex / miRNA-mediated gene silencing by mRNA destabilization / regulation of stem cell population maintenance / SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins ...positive regulation of cytoplasmic mRNA processing body assembly / poly(A)-specific ribonuclease / poly(A)-specific ribonuclease activity / CCR4-NOT core complex / armadillo repeat domain binding / CCR4-NOT complex / miRNA-mediated gene silencing by mRNA destabilization / regulation of stem cell population maintenance / SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / SUMO is proteolytically processed / positive regulation of mRNA catabolic process / negative regulation of retinoic acid receptor signaling pathway / SUMOylation of transcription factors / SUMOylation of transcription cofactors / Postmitotic nuclear pore complex (NPC) reformation / nuclear-transcribed mRNA poly(A) tail shortening / septin ring / SUMOylation of DNA damage response and repair proteins / negative regulation of intracellular estrogen receptor signaling pathway / Transcriptional and post-translational regulation of MITF-M expression and activity / SUMOylation of DNA replication proteins / trophectodermal cell differentiation / miRNA-mediated post-transcriptional gene silencing / Deadenylation of mRNA / nuclear retinoic acid receptor binding / SUMOylation of SUMOylation proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / M-decay: degradation of maternal mRNAs by maternally stored factors / SUMOylation of RNA binding proteins / SUMOylation of chromatin organization proteins / peroxisomal membrane / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / TP53 regulates transcription of additional cell cycle genes whose exact role in the p53 pathway remain uncertain / positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / ubiquitin-like protein ligase binding / protein sumoylation / condensed nuclear chromosome / nuclear estrogen receptor binding / P-body / protein tag activity / regulation of translation / 3'-5'-RNA exonuclease activity / molecular adaptor activity / negative regulation of translation / protein domain specific binding / positive regulation of cell population proliferation / negative regulation of transcription by RNA polymerase II / extracellular space / RNA binding / metal ion binding / identical protein binding / nucleus / membrane / cytosol
Similarity search - Function
CCR4-NOT transcription complex subunit 7/8/Pop2 / Ribonuclease CAF1 / CAF1 family ribonuclease / : / CCR4-NOT transcription complex subunit 1, N-terminal domain / CCR4-NOT transcription complex subunit 1, NOT1_connector / : / CCR4-Not complex component, Not1, C-terminal / CCR4-NOT transcription complex subunit 1, domain 4 / CCR4-NOT transcription complex subunit 1, CAF1-binding domain ...CCR4-NOT transcription complex subunit 7/8/Pop2 / Ribonuclease CAF1 / CAF1 family ribonuclease / : / CCR4-NOT transcription complex subunit 1, N-terminal domain / CCR4-NOT transcription complex subunit 1, NOT1_connector / : / CCR4-Not complex component, Not1, C-terminal / CCR4-NOT transcription complex subunit 1, domain 4 / CCR4-NOT transcription complex subunit 1, CAF1-binding domain / CCR4-NOT transcription complex subunit 1, TTP binding domain / CCR4-NOT transcription complex subunit 1, HEAT repeat / CCR4-NOT subunit 1, TTP binding domain superfamily / CCR4-NOT transcription complex subunit 1 / CCR4-Not complex component, Not1 / CCR4-Not complex, Not1 subunit, domain of unknown function DUF3819 / CCR4-NOT transcription complex subunit 1 CAF1-binding domain / CCR4-NOT transcription complex subunit 1 TTP binding domain / CCR4-NOT transcription complex subunit 1 HEAT repeat / Rad60/SUMO-like domain / Ubiquitin-2 like Rad60 SUMO-like / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Dot/Icm T4SS effector PieF / CCR4-NOT transcription complex subunit 1 / Ubiquitin-like protein SMT3 / CCR4-NOT transcription complex subunit 8
Similarity search - Component
Biological speciesHomo sapiens (human) / Legionella pneumophila (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLevdansky E / Deme J / Lea SM / Valkov E
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)Intramural Research Program United States
CitationJournal: Nat Commun / Year: 2025
Title: Intracellular pathogen effector reprograms host gene expression by inhibiting mRNA decay.
Authors: Yevgen Levdansky / Justin C Deme / David J Turner / Claire T Piczak / Filip Pekovic / Anna L Valkov / Sergey G Tarasov / Susan M Lea / Eugene Valkov /
Abstract: Legionella pneumophila, an intracellular bacterial pathogen, injects effector proteins into host cells to manipulate cellular processes and promote its survival and proliferation. Here, we reveal a ...Legionella pneumophila, an intracellular bacterial pathogen, injects effector proteins into host cells to manipulate cellular processes and promote its survival and proliferation. Here, we reveal a unique mechanism by which the Legionella effector PieF perturbs host mRNA decay by targeting the human CCR4-NOT deadenylase complex. High-resolution cryo-electron microscopy structures and biochemical analyses reveal that PieF binds with nanomolar affinity to the NOT7 and NOT8 catalytic subunits of CCR4-NOT, obstructing RNA access and displacing a catalytic Mg²⁺ ion from the active site. Additionally, PieF prevents NOT7/8 from associating with their partner deadenylases NOT6/6L, inhibiting the assembly of a functional deadenylase complex. Consequently, PieF robustly blocks mRNA poly(A) tail shortening and degradation with striking potency and selectivity for NOT7/8. This inhibition of deadenylation by PieF impedes cell cycle progression in human cells, revealing a novel bacterial strategy to modulate host gene expression.
History
DepositionNov 4, 2024-
Header (metadata) releaseJul 23, 2025-
Map releaseJul 23, 2025-
UpdateJul 23, 2025-
Current statusJul 23, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47690.png

Downloads & links

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Map

FileDownload / File: emd_47690.map.gz / Format: CCP4 / Size: 229.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap used for model refinement
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 392 pix.
= 286.944 Å		0.73 Å/pix.
x 392 pix.
= 286.944 Å		0.73 Å/pix.
x 392 pix.
= 286.944 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.732 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.0018131735 - 1.829546
Average (Standard dev.)0.00045388896 (±0.014956891)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions392392392
Spacing392392392
CellA=B=C: 286.944 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47690_msk_1.map
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Additional map: mask used for FSC calculation

Fileemd_47690_additional_1.map
Annotationmask used for FSC calculation
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Additional map: B-factor sharpened map

Fileemd_47690_additional_2.map
AnnotationB-factor sharpened map
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Additional map: unsharpened map

Fileemd_47690_additional_3.map
Annotationunsharpened map
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Half map: half map 1

Fileemd_47690_half_map_1.map
Annotationhalf map 1
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Half map: half map 2

Fileemd_47690_half_map_2.map
Annotationhalf map 2
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Sample components

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Entire : NOT1:NOT8:PieF

EntireName: NOT1:NOT8:PieF
Components
  • Complex: NOT1:NOT8:PieF
    • Protein or peptide: Ubiquitin-like protein SMT3,CCR4-NOT transcription complex subunit 8
    • Protein or peptide: Dot/Icm T4SS effector PieF
    • Protein or peptide: CCR4-NOT transcription complex subunit 1
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: NOT1:NOT8:PieF

SupramoleculeName: NOT1:NOT8:PieF / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Ubiquitin-like protein SMT3,CCR4-NOT transcription complex subunit 8

MacromoleculeName: Ubiquitin-like protein SMT3,CCR4-NOT transcription complex subunit 8
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: poly(A)-specific ribonuclease
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.754742 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGTMSDSEV NQEAKPEVKP EVKPETHINL KVSDGSSEIF FKIKKTTPLR RLMEAFAKRQ GKEMDSLRFL YDGIRIQAD QTPEDLDMED NDIIEAHREQ TGGSTGSGLE VLFQGPHMPA ALVENSQVIC EVWASNLEEE MRKIREIVLS Y SYIAMDTE ...String:
MGSSHHHHHH SSGTMSDSEV NQEAKPEVKP EVKPETHINL KVSDGSSEIF FKIKKTTPLR RLMEAFAKRQ GKEMDSLRFL YDGIRIQAD QTPEDLDMED NDIIEAHREQ TGGSTGSGLE VLFQGPHMPA ALVENSQVIC EVWASNLEEE MRKIREIVLS Y SYIAMDTE FPGVVVRPIG EFRSSIDYQY QLLRCNVDLL KIIQLGLTFT NEKGEYPSGI NTWQFNFKFN LTEDMYSQDS ID LLANSGL QFQKHEEEGI DTLHFAELLM TSGVVLCDNV KWLSFHSGYD FGYMVKLLTD SRLPEEEHEF FHILNLFFPS IYD VKYLMK SCKNLKGGLQ EVADQLDLQR IGRQHQAGSD SLLTGMAFFR MKELFFEDSI DDAKYCGRLY GLGTGVAQKQ NEDV DSAQE KMSILAIINN MQQ

UniProtKB: Ubiquitin-like protein SMT3, CCR4-NOT transcription complex subunit 8

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Macromolecule #2: Dot/Icm T4SS effector PieF

MacromoleculeName: Dot/Icm T4SS effector PieF / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila (bacteria)
Molecular weightTheoretical: 16.568549 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGSSHHHHHH SSGTGSGENL YFQGHMKRLI ICNGNKLTVC TQAISSGGIV EKYTPIFSLT KESDNELTLE LSGVARGYYI IPSELTSSQ ARAAHLITLL TRAEESQTTD MHKILNSFVS GKITSGSMFN FENDGSFKRE PEEAYNLINK I

UniProtKB: Dot/Icm T4SS effector PieF

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Macromolecule #3: CCR4-NOT transcription complex subunit 1

MacromoleculeName: CCR4-NOT transcription complex subunit 1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 29.237787 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGTGSGLEV LFQGPHMLEE NIQEKIAFIF NNLSQSNMTQ KVEELKETVK EEFMPWVSQY LVMKRVSIEP NFHSLYSNF LDTLKNPEFN KMVLNETYRN IKVLLTSDKA AANFSDRSLL KNLGHWLGMI TLAKNKPILH TDLDVKSLLL E AYVKGQQE ...String:
MGSSHHHHHH SSGTGSGLEV LFQGPHMLEE NIQEKIAFIF NNLSQSNMTQ KVEELKETVK EEFMPWVSQY LVMKRVSIEP NFHSLYSNF LDTLKNPEFN KMVLNETYRN IKVLLTSDKA AANFSDRSLL KNLGHWLGMI TLAKNKPILH TDLDVKSLLL E AYVKGQQE LLYVVPFVAK VLESSIRSVV FRPPNPWTMA IMNVLAELHQ EHDLKLNLKF EIEVLCKNLA LDINELKPGN LL KDKDRLK NLDEQLS

UniProtKB: CCR4-NOT transcription complex subunit 1

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 52.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 200675
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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