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- PDB-9e7u: Cryo-EM structure of NOT1:NOT8:PieF -

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Basic information

Entry
Database: PDB / ID: 9e7u
TitleCryo-EM structure of NOT1:NOT8:PieF
Components
  • CCR4-NOT transcription complex subunit 1
  • Dot/Icm T4SS effector PieF
  • Ubiquitin-like protein SMT3,CCR4-NOT transcription complex subunit 8
KeywordsGENE REGULATION / PieF / mRNA decay / deadenylation / CCR4-NOT / host-pathogen interactions
Function / homology
Function and homology information


positive regulation of cytoplasmic mRNA processing body assembly / poly(A)-specific ribonuclease / poly(A)-specific ribonuclease activity / armadillo repeat domain binding / CCR4-NOT core complex / CCR4-NOT complex / miRNA-mediated gene silencing by mRNA destabilization / regulation of stem cell population maintenance / positive regulation of mRNA catabolic process / SUMO is conjugated to E1 (UBA2:SAE1) ...positive regulation of cytoplasmic mRNA processing body assembly / poly(A)-specific ribonuclease / poly(A)-specific ribonuclease activity / armadillo repeat domain binding / CCR4-NOT core complex / CCR4-NOT complex / miRNA-mediated gene silencing by mRNA destabilization / regulation of stem cell population maintenance / positive regulation of mRNA catabolic process / SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / negative regulation of retinoic acid receptor signaling pathway / SUMO is proteolytically processed / nuclear-transcribed mRNA poly(A) tail shortening / SUMOylation of transcription factors / SUMOylation of transcription cofactors / Postmitotic nuclear pore complex (NPC) reformation / 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 / trophectodermal cell differentiation / miRNA-mediated post-transcriptional gene silencing / SUMOylation of DNA replication proteins / Deadenylation of mRNA / nuclear retinoic acid receptor binding / SUMOylation of SUMOylation proteins / M-decay: degradation of maternal mRNAs by maternally stored factors / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / SUMOylation of RNA binding proteins / peroxisomal membrane / SUMOylation of chromatin organization proteins / 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 / nuclear estrogen receptor binding / condensed nuclear chromosome / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLevdansky, E. / Deme, J. / Lea, S.M. / Valkov, E.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 23, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Ubiquitin-like protein SMT3,CCR4-NOT transcription complex subunit 8
A: Dot/Icm T4SS effector PieF
B: CCR4-NOT transcription complex subunit 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)93,5854
Polymers93,5613
Non-polymers241
Water362
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, confirmed by mass photometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Ubiquitin-like protein SMT3,CCR4-NOT transcription complex subunit 8 / CAF1-like protein / CALIFp / CAF2 / CCR4-associated factor 8 / Caf1b


Mass: 47754.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SMT3, YDR510W, D9719.15, CNOT8, CALIF, POP2 / Production host: Escherichia coli (E. coli)
References: UniProt: Q12306, UniProt: Q9UFF9, poly(A)-specific ribonuclease
#2: Protein Dot/Icm T4SS effector PieF


Mass: 16568.549 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Legionella pneumophila (bacteria) / Gene: pieF, JBJ86_12865 / Production host: Escherichia coli (E. coli) / References: UniProt: A0AAN5PHN6
#3: Protein CCR4-NOT transcription complex subunit 1 / CCR4-associated factor 1 / Negative regulator of transcription subunit 1 homolog / NOT1H / hNOT1


Mass: 29237.787 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CNOT1, CDC39, KIAA1007, NOT1, AD-005 / Production host: Escherichia coli (E. coli) / References: UniProt: A5YKK6
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
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: NOT1:NOT8:PieF / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 52.4 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 200675 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0054956
ELECTRON MICROSCOPYf_angle_d0.6216686
ELECTRON MICROSCOPYf_dihedral_angle_d6.63649
ELECTRON MICROSCOPYf_chiral_restr0.041751
ELECTRON MICROSCOPYf_plane_restr0.004854

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