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- EMDB-47689: Cryo-EM structure of NOT1:NOT7:PieF -

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Basic information

Entry
Database: EMDB / ID: EMD-47689
TitleCryo-EM structure of NOT1:NOT7:PieF
Map datasharpened map
Sample
  • Complex: NOT1:NOT7:PieF
    • Protein or peptide: Dot/Icm T4SS effector PieF
    • Protein or peptide: CCR4-NOT transcription complex subunit 1
    • Protein or peptide: SDH7p Mitochondrial protein involved in assembly of succinate dehydrogenase,CCR4-NOT transcription complex subunit 7
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsPieF / mRNA decay / deadenylation / CCR4-NOT / host-pathogen interactions / GENE REGULATION
Function / homology
Function and homology information


mitochondrial respiratory chain complex II assembly / regulation of tyrosine phosphorylation of STAT protein / positive regulation of cytoplasmic mRNA processing body assembly / poly(A)-specific ribonuclease / piRNA binding / piRNA-mediated gene silencing by mRNA destabilization / poly(A)-specific ribonuclease activity / RNA exonuclease activity / CCR4-NOT core complex / armadillo repeat domain binding ...mitochondrial respiratory chain complex II assembly / regulation of tyrosine phosphorylation of STAT protein / positive regulation of cytoplasmic mRNA processing body assembly / poly(A)-specific ribonuclease / piRNA binding / piRNA-mediated gene silencing by mRNA destabilization / poly(A)-specific ribonuclease activity / RNA exonuclease activity / CCR4-NOT core complex / armadillo repeat domain binding / CCR4-NOT complex / succinate metabolic process / miRNA-mediated gene silencing by mRNA destabilization / regulation of stem cell population maintenance / positive regulation of mRNA catabolic process / negative regulation of retinoic acid receptor signaling pathway / nuclear-transcribed mRNA poly(A) tail shortening / deadenylation-dependent decapping of nuclear-transcribed mRNA / negative regulation of intracellular estrogen receptor signaling pathway / trophectodermal cell differentiation / miRNA-mediated post-transcriptional gene silencing / Deadenylation of mRNA / nuclear retinoic acid receptor binding / M-decay: degradation of maternal mRNAs by maternally stored factors / regulatory ncRNA-mediated gene silencing / peroxisomal membrane / P-body assembly / 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 / negative regulation of type I interferon-mediated signaling pathway / intracellular membraneless organelle / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / positive regulation of viral genome replication / nuclear estrogen receptor binding / P-body / mitochondrial intermembrane space / transcription corepressor activity / regulation of translation / 3'-5'-RNA exonuclease activity / defense response to virus / molecular adaptor activity / DNA-binding transcription factor binding / negative regulation of translation / nuclear speck / nuclear body / mitochondrial matrix / protein domain specific binding / negative regulation of cell population proliferation / negative regulation of gene expression / negative regulation of DNA-templated transcription / positive regulation of cell population proliferation / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / extracellular space / RNA binding / metal ion binding / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
Succinate dehydrogenase assembly factor 3, mitochondrial / 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 ...Succinate dehydrogenase assembly factor 3, mitochondrial / 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 / Complex1_LYR-like / 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
Succinate dehydrogenase assembly factor 3 / Dot/Icm T4SS effector PieF / CCR4-NOT transcription complex subunit 1 / CCR4-NOT transcription complex subunit 7
Similarity search - Component
Biological speciesHomo sapiens (human) / Legionella pneumophila (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 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_47689.png

Downloads & links

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Map

FileDownload / File: emd_47689.map.gz / Format: CCP4 / Size: 229.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
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

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.732 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-2.128472 - 2.6499536
Average (Standard dev.)0.00022693719 (±0.03660786)
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_47689_msk_1.map
Projections & Slices
AxesZYX

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Histogram

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Additional map: unsharpened map

Fileemd_47689_additional_1.map
Annotationunsharpened map
Projections & Slices
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Half map: half map 2

Fileemd_47689_half_map_1.map
Annotationhalf map 2
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_47689_half_map_2.map
Annotationhalf map 1
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Histogram

Histogram (log scale)

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Sample components

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Entire : NOT1:NOT7:PieF

EntireName: NOT1:NOT7:PieF
Components
  • Complex: NOT1:NOT7:PieF
    • Protein or peptide: Dot/Icm T4SS effector PieF
    • Protein or peptide: CCR4-NOT transcription complex subunit 1
    • Protein or peptide: SDH7p Mitochondrial protein involved in assembly of succinate dehydrogenase,CCR4-NOT transcription complex subunit 7
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: NOT1:NOT7:PieF

SupramoleculeName: NOT1:NOT7:PieF / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Dot/Icm T4SS effector PieF

MacromoleculeName: Dot/Icm T4SS effector PieF / type: protein_or_peptide / ID: 1 / 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 #2: CCR4-NOT transcription complex subunit 1

MacromoleculeName: CCR4-NOT transcription complex subunit 1 / type: protein_or_peptide / ID: 2 / 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 #3: SDH7p Mitochondrial protein involved in assembly of succinate deh...

MacromoleculeName: SDH7p Mitochondrial protein involved in assembly of succinate dehydrogenase,CCR4-NOT transcription complex subunit 7
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: poly(A)-specific ribonuclease
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.134922 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MSDSEVNQEA KPEVKPEVKP ETHINLKVSD GSSEIFFKIK KTTPLRRLME AFAKRQGKEM DSLRFLYDG IRIQADQTPE DLDMEDNDII EAHREQTGGS ENLYFQGMPA ATVDHSQRIC EVWACNLDEE MKKIRQVIRK Y NYVAMDTE ...String:
MGSSHHHHHH SSGLVPRGSH MSDSEVNQEA KPEVKPEVKP ETHINLKVSD GSSEIFFKIK KTTPLRRLME AFAKRQGKEM DSLRFLYDG IRIQADQTPE DLDMEDNDII EAHREQTGGS ENLYFQGMPA ATVDHSQRIC EVWACNLDEE MKKIRQVIRK Y NYVAMDTE FPGVVARPIG EFRSNADYQY QLLRCNVDLL KIIQLGLTFM NEQGEYPPGT STWQFNFKFN LTEDMYAQDS IE LLTTSGI QFKKHEEEGI ETQYFAELLM TSGVVLCEGV KWLSFHSGYD FGYLIKILTN SNLPEEELDF FEILRLFFPV IYD VKYLMK SCKNLKGGLQ EVAEQLELER IGPQHQAGSD SLLTGMAFFK MREMFFEDHI DDAKYCGHLY GLGSGSSYVQ NGTG NAYEE EANKQS

UniProtKB: Succinate dehydrogenase assembly factor 3, CCR4-NOT transcription complex subunit 7

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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: 55.6 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: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 417581
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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