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- PDB-9qdq: Cryo-EM structure of Upf1-Nmd4-Ebs1 in complex with RNA -

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Basic information

Entry
Database: PDB / ID: 9qdq
TitleCryo-EM structure of Upf1-Nmd4-Ebs1 in complex with RNA
Components
  • ATP-dependent helicase NAM7
  • Nonsense-mediated decay protein 4
  • Nonsense-mediated mRNA decay factor EBS1
  • RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')
KeywordsRNA BINDING PROTEIN / helicase / Nonsense-mediated decay
Function / homology
Function and homology information


cytoplasmic RNA surveillance / double-stranded DNA helicase activity / nuclear-transcribed mRNA catabolic process, 3'-5' exonucleolytic nonsense-mediated decay / regulation of translational termination / intracellular mRNA localization / telomerase holoenzyme complex / telomerase RNA binding / silent mating-type cassette heterochromatin formation / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / telomeric DNA binding ...cytoplasmic RNA surveillance / double-stranded DNA helicase activity / nuclear-transcribed mRNA catabolic process, 3'-5' exonucleolytic nonsense-mediated decay / regulation of translational termination / intracellular mRNA localization / telomerase holoenzyme complex / telomerase RNA binding / silent mating-type cassette heterochromatin formation / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / telomeric DNA binding / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / nuclear-transcribed mRNA catabolic process / ribosomal small subunit binding / P-body / single-stranded DNA binding / DNA recombination / DNA helicase / chromosome, telomeric region / RNA helicase activity / negative regulation of translation / protein ubiquitination / RNA helicase / mRNA binding / ATP hydrolysis activity / RNA binding / zinc ion binding / ATP binding / nucleus / cytoplasm
Similarity search - Function
Telomerase activating protein Est1-like, N-terminal / Telomerase activating protein Est1 / DNA/RNA-binding domain, Est1-type / Est1/Ebs1-like / Est1 DNA/RNA binding domain / RNA helicase UPF1, 1B domain / RNA helicase (UPF2 interacting domain) / RNA helicase UPF1, 1B domain / Upf1 cysteine-histidine-rich (CH-rich) domain profile. / RNA helicase UPF1, Cys/His rich zinc-binding domain ...Telomerase activating protein Est1-like, N-terminal / Telomerase activating protein Est1 / DNA/RNA-binding domain, Est1-type / Est1/Ebs1-like / Est1 DNA/RNA binding domain / RNA helicase UPF1, 1B domain / RNA helicase (UPF2 interacting domain) / RNA helicase UPF1, 1B domain / Upf1 cysteine-histidine-rich (CH-rich) domain profile. / RNA helicase UPF1, Cys/His rich zinc-binding domain / DNA2/NAM7 helicase, helicase domain / AAA domain / DNA2/NAM7-like helicase / : / Large family of predicted nucleotide-binding domains / PIN domain / Tetratricopeptide-like helical domain superfamily / DNA2/NAM7 helicase-like, C-terminal / AAA domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA / RNA (> 10) / ATP-dependent helicase NAM7 / Nonsense-mediated mRNA decay factor EBS1 / Nonsense-mediated decay protein 4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsIermak, I. / Wilson Eisele, N.R. / Kurscheidt, K. / Loukeri, M.J. / Basquin, J. / Bonneau, F. / Langer, L.M. / Keidel, A. / Conti, E.
Funding support Germany, European Union, Denmark, 5items
OrganizationGrant numberCountry
Max Planck Society Germany
European Research Council (ERC)740329European Union
European Research Council (ERC)101054447European Union
German Research Foundation (DFG)SFB1035 Germany
Novo Nordisk Foundation31199 Denmark
CitationJournal: Cell Rep / Year: 2025
Title: Molecular mechanisms governing the formation of distinct Upf1-containing complexes in yeast.
Authors: Iuliia Iermak / Nicole R Wilson Eisele / Katharina Kurscheidt / Matina-Jasemi Loukeri / Jérôme Basquin / Fabien Bonneau / Lukas M Langer / Achim Keidel / Elena Conti /
Abstract: Upf1 is a master regulator of nonsense-mediated mRNA decay (NMD), an mRNA surveillance and degradation pathway conserved from yeast to human. In Saccharomyces cerevisiae, Upf1 exists in two distinct ...Upf1 is a master regulator of nonsense-mediated mRNA decay (NMD), an mRNA surveillance and degradation pathway conserved from yeast to human. In Saccharomyces cerevisiae, Upf1 exists in two distinct complexes with factors that mediate NMD activation or 5'-3' mRNA degradation. We combined endogenous purifications and biochemical reconstitutions of yeast Upf1 complexes with structural analyses and biochemical assays to elucidate the molecular mechanisms driving the organization of the Upf1-5'-3' and Upf1-2-3 complexes. We show that yeast Upf1 is in a constitutive complex, whereby its CH, RecA, and C-terminal domains interact with the mRNA decapping factor Dcp2, NMD-associated proteins Nmd4 and Ebs1, and the 5'-3' exoribonuclease Xrn1, respectively. Together, the interacting surfaces and closed conformation of Upf1 in the Upf1-5'-3' complex sterically obstruct the binding of Upf2-3. Our work points to a major restructuring upon recruitment of these factors during NMD and provides insights into evolutionary divergence amongst species.
History
DepositionMar 6, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 22, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ATP-dependent helicase NAM7
B: Nonsense-mediated mRNA decay factor EBS1
C: Nonsense-mediated decay protein 4
D: RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')


Theoretical massNumber of molelcules
Total (without water)244,1204
Polymers244,1204
Non-polymers00
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 ATP-dependent helicase NAM7 / Nonsense-mediated mRNA decay protein 1 / Nuclear accommodation of mitochondria 7 protein / Up- ...Nonsense-mediated mRNA decay protein 1 / Nuclear accommodation of mitochondria 7 protein / Up-frameshift suppressor 1


Mass: 109561.008 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: NAM7, IFS2, MOF4, UPF1, YMR080C, YM9582.05C / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P30771, DNA helicase, RNA helicase
#2: Protein Nonsense-mediated mRNA decay factor EBS1 / EST1-like BCY1 suppressor 1


Mass: 100102.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: EBS1, YDR206W, YD8142.03 / Production host: Escherichia coli (E. coli) / References: UniProt: Q03466
#3: Protein Nonsense-mediated decay protein 4


Mass: 25316.912 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: NMD4, YLR363C / Production host: Escherichia coli (E. coli) / References: UniProt: Q12129
#4: RNA chain RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')


Mass: 9140.017 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
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: Complex of Upf1, Nmd4 and Ebs1 bound to RNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE-PROPANE

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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: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
7UCSF ChimeraXmodel fitting
13PHENIX1.20.1_4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 160049 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Initial fitting of the Alphafold3 model was done in ChimeraX. Then model was rigid body refined and real space refined in Phenix
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.6 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311633
ELECTRON MICROSCOPYf_angle_d0.75215773
ELECTRON MICROSCOPYf_dihedral_angle_d15.0664370
ELECTRON MICROSCOPYf_chiral_restr0.0451784
ELECTRON MICROSCOPYf_plane_restr0.0051984

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