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- EMDB-53032: Cryo-EM structure of Upf1-Nmd4-Ebs1 in complex with RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-53032
TitleCryo-EM structure of Upf1-Nmd4-Ebs1 in complex with RNA
Map data
Sample
  • Complex: Complex of Upf1, Nmd4 and Ebs1 bound to RNA
    • Protein or peptide: ATP-dependent helicase NAM7
    • Protein or peptide: Nonsense-mediated mRNA decay factor EBS1
    • Protein or peptide: Nonsense-mediated decay protein 4
    • RNA: RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')
Keywordshelicase / Nonsense-mediated decay / RNA BINDING PROTEIN
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 / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / silent mating-type cassette heterochromatin formation / 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 / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / silent mating-type cassette heterochromatin formation / 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 / Large family of predicted nucleotide-binding domains / DNA2/NAM7-like helicase / : / 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
ATP-dependent helicase NAM7 / Nonsense-mediated mRNA decay factor EBS1 / Nonsense-mediated decay protein 4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsIermak I / Wilson Eisele NR / Kurscheidt K / Loukeri MJ / Basquin J / Bonneau F / Langer LM / Keidel A / Conti E
Funding support Germany, European Union, Denmark, 5 items
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, 2025-
Header (metadata) releaseOct 22, 2025-
Map releaseOct 22, 2025-
UpdateOct 22, 2025-
Current statusOct 22, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53032.png

Downloads & links

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Map

FileDownload / File: emd_53032.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 360 pix.
= 306.432 Å		0.85 Å/pix.
x 360 pix.
= 306.432 Å		0.85 Å/pix.
x 360 pix.
= 306.432 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8512 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.1890716 - 0.35995764
Average (Standard dev.)0.000116898715 (±0.0086842915)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 306.432 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_53032_msk_1.map
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Additional map: #2

Fileemd_53032_additional_1.map
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Additional map: #1

Fileemd_53032_additional_2.map
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Half map: #2

Fileemd_53032_half_map_1.map
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Half map: #1

Fileemd_53032_half_map_2.map
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Sample components

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Entire : Complex of Upf1, Nmd4 and Ebs1 bound to RNA

EntireName: Complex of Upf1, Nmd4 and Ebs1 bound to RNA
Components
  • Complex: Complex of Upf1, Nmd4 and Ebs1 bound to RNA
    • Protein or peptide: ATP-dependent helicase NAM7
    • Protein or peptide: Nonsense-mediated mRNA decay factor EBS1
    • Protein or peptide: Nonsense-mediated decay protein 4
    • RNA: RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')

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Supramolecule #1: Complex of Upf1, Nmd4 and Ebs1 bound to RNA

SupramoleculeName: Complex of Upf1, Nmd4 and Ebs1 bound to RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: ATP-dependent helicase NAM7

MacromoleculeName: ATP-dependent helicase NAM7 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 109.561008 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MVGSGSHTPY DISNSPSDVN VQPATQLNST LVEDDDVDNQ LFEEAQVTET GFRSPSASDN SCAYCGIDSA KCVIKCNSCK KWFCNTKNG TSSSHIVNHL VLSHHNVVSL HPDSDLGDTV LECYNCGRKN VFLLGFVSAK SEAVVVLLCR IPCAQTKNAN W DTDQWQPL ...String:
MVGSGSHTPY DISNSPSDVN VQPATQLNST LVEDDDVDNQ LFEEAQVTET GFRSPSASDN SCAYCGIDSA KCVIKCNSCK KWFCNTKNG TSSSHIVNHL VLSHHNVVSL HPDSDLGDTV LECYNCGRKN VFLLGFVSAK SEAVVVLLCR IPCAQTKNAN W DTDQWQPL IEDRQLLSWV AEQPTEEEKL KARLITPSQI SKLEAKWRSN KDATINDIDA PEEQEAIPPL LLRYQDAYEY QR SYGPLIK LEADYDKQLK ESQALEHISV SWSLALNNRH LASFTLSTFE SNELKVAIGD EMILWYSGMQ HPDWEGRGYI VRL PNSFQD TFTLELKPSK TPPPTHLTTG FTAEFIWKGT SYDRMQDALK KFAIDKKSIS GYLYYKILGH QVVDISFDVP LPKE FSIPN FAQLNSSQSN AVSHVLQRPL SLIQGPPGTG KTVTSATIVY HLSKIHKDRI LVCAPSNVAV DHLAAKLRDL GLKVV RLTA KSREDVESSV SNLALHNLVG RGAKGELKNL LKLKDEVGEL SASDTKRFVK LVRKTEAEIL NKADVVCCTC VGAGDK RLD TKFRTVLIDE STQASEPECL IPIVKGAKQV ILVGDHQQLG PVILERKAAD AGLKQSLFER LISLGHVPIR LEVQYRM NP YLSEFPSNMF YEGSLQNGVT IEQRTVPNSK FPWPIRGIPM MFWANYGREE ISANGTSFLN RIEAMNCERI ITKLFRDG V KPEQIGVITP YEGQRAYILQ YMQMNGSLDK DLYIKVEVAS VDAFQGREKD YIILSCVRAN EQQAIGFLRD PRRLNVGLT RAKYGLVILG NPRSLARNTL WNHLLIHFRE KGCLVEGTLD NLQLCTVQLV RPQPRKTERP MNAQFNVESE MGDFPKFQDF DAQSMVSFS GQIGDFGNAF VDNTELSSYI NNEYWNFENF KSAFSQKQNR NEIDDRNLYQ EEASHLNSNF ARELQREEQK H ELSKDFSN LGI

UniProtKB: ATP-dependent helicase NAM7

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Macromolecule #2: Nonsense-mediated mRNA decay factor EBS1

MacromoleculeName: Nonsense-mediated mRNA decay factor EBS1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 100.102453 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEPSNTQKED LPTAFNGIKS QLNSILKSNQ LFQDYALLNG FLAFVHSKLN AAILTSIESQ CGKSFAADLD SFDQSSISSI LDFSWESVH YPIFKWFQMW RNYILFEKEN KKQQTKFIDF RKMNSKMLKF FKTVQNFYVN VINTVYKKYD ISVLLPKRII Q DLKLSDIE ...String:
MEPSNTQKED LPTAFNGIKS QLNSILKSNQ LFQDYALLNG FLAFVHSKLN AAILTSIESQ CGKSFAADLD SFDQSSISSI LDFSWESVH YPIFKWFQMW RNYILFEKEN KKQQTKFIDF RKMNSKMLKF FKTVQNFYVN VINTVYKKYD ISVLLPKRII Q DLKLSDIE NTTNVGDILA VKTFNSSSPL AHLIPTLFHR CLLFLGTAYR YKTLLEEISN KYSISNFKKS LDFFRLASLV LP SAGETYS QAGAIFLQTG NLGIAVFNFV KGMMTKMPSP VSIKNFGALM VDNKSSLNRS LHTTIMNTYL QESKGPRTPA KEI LEFYFL GLFGSVWSPT SWRDDTKPNQ LNNGIKLRHL ENALYETMSA RYLNNIKTIF HNLIITIGGF HLLLKRRSDV SAKT LKDLR SNELDYLNFA FKYIAHILND IVKESWSENP EVSEILGMVR IINCWIKANP MVLQYSQSNL EFVNALAYLI NDIVK KKPS PSFSITEHIP KRTYWFEEDL MVKGLSFVNF QLSDFDDYEK ILEMDHSLDR LIGNPPLCDK LSASSEMLLR LQAVVN ISS QLLQNNNCGV EWSDNKSRYI FNKKIGFKET VKNSMKTSKQ SNEKAKLQRK NKPSTTNGSI SMADLERQMR SSSLDSF SP TMGYSGSSVP MAPDTFNVKP SGTITGNKVN VELLKIELSG QNADGAITNI SPGYSNAAIS SSNSTDESSF DLNNILSS M QQKHAEKSFA KSMQGVNEQI PANDVCHQAQ RPMQGGLYSS QQPSSMSSLN SAYQNASMPP SASMVSYPYP FLNQQGQGV FPPYNAQNLQ WQSEAYSLKS MNFANPTWLG DQYQTSAPSS AYAQAQRQMF QQPMQQDVGK YMQFPFDAQS NTDSMRGNSR NNMF

UniProtKB: Nonsense-mediated mRNA decay factor EBS1

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Macromolecule #3: Nonsense-mediated decay protein 4

MacromoleculeName: Nonsense-mediated decay protein 4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 25.316912 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTQYNFIIDA SAFEKGLGNI KRWCSDCTEA VTLNFYIPTF TLNELDFLQQ RRKSFAARES LKFIDRLDDS KFANLKVFIE FPEVLDIIL WSDVMEHNDS SGKINIAKLP KRLKNLLKSC IYKCYLEGNE GLHWFLISED PQIREMAMQC NIPSCSIVDV D SILSKDMN ...String:
MTQYNFIIDA SAFEKGLGNI KRWCSDCTEA VTLNFYIPTF TLNELDFLQQ RRKSFAARES LKFIDRLDDS KFANLKVFIE FPEVLDIIL WSDVMEHNDS SGKINIAKLP KRLKNLLKSC IYKCYLEGNE GLHWFLISED PQIREMAMQC NIPSCSIVDV D SILSKDMN DKSFRESEKF NNMMLKNGTK EESENGREII RTNFNKTVYA SRGTGELWSP

UniProtKB: Nonsense-mediated decay protein 4

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Macromolecule #4: RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')

MacromoleculeName: RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3') / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 9.140017 KDa
SequenceString:
UUUUUUUUUU UUUUUUUUUU UUUUUUUUUU

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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
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 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: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 160049
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsInitial fitting of the Alphafold3 model was done in ChimeraX. Then model was rigid body refined and real space refined in Phenix
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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