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- EMDB-19451: Human UPF1 RNA helicase with AMPPNP and RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-19451
TitleHuman UPF1 RNA helicase with AMPPNP and RNA
Map dataHuman UPF1 bound to AMPPNP and RNA
Sample
  • Complex: UPF1 bound to AMPPNP
    • Protein or peptide: UPF1, RENT1
KeywordsRNA helicase / human / nonsense-mediated mRNA decay / RNA BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.51 Å
AuthorsLanger LM / Conti E
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nucleic Acids Res / Year: 2024
Title: UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2.
Authors: Lukas M Langer / Katharina Kurscheidt / Jérôme Basquin / Fabien Bonneau / Iuliia Iermak / Claire Basquin / Elena Conti /
Abstract: Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the ...Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.
History
DepositionJan 20, 2024-
Header (metadata) releaseMay 15, 2024-
Map releaseMay 15, 2024-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19451.png

Downloads & links

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Map

FileDownload / File: emd_19451.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman UPF1 bound to AMPPNP and RNA
Voxel sizeX=Y=Z: 1.885 Å
Density
Contour LevelBy AUTHOR: 0.944
Minimum - Maximum-2.631954 - 5.0467634
Average (Standard dev.)-0.0044289846 (±0.13916375)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 241.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_19451_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_19451_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : UPF1 bound to AMPPNP

EntireName: UPF1 bound to AMPPNP
Components
  • Complex: UPF1 bound to AMPPNP
    • Protein or peptide: UPF1, RENT1

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Supramolecule #1: UPF1 bound to AMPPNP

SupramoleculeName: UPF1 bound to AMPPNP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 124 KDa

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Macromolecule #1: UPF1, RENT1

MacromoleculeName: UPF1, RENT1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSVEAYGPSS QTLTFLDTEE AELLGADTQG SEFEFTDFTL PSQTQTPPGG PGGPGGGGAG GPGGAGAGAA AGQLDAQVGP EGILQNGAVD DSVAKTSQLL AELNFEEDEE DTYYTKDLPI HACSYCGIHD PACVVYCNTS KKWFCNGRGN TSGSHIVNHL VRAKCKEVTL ...String:
MSVEAYGPSS QTLTFLDTEE AELLGADTQG SEFEFTDFTL PSQTQTPPGG PGGPGGGGAG GPGGAGAGAA AGQLDAQVGP EGILQNGAVD DSVAKTSQLL AELNFEEDEE DTYYTKDLPI HACSYCGIHD PACVVYCNTS KKWFCNGRGN TSGSHIVNHL VRAKCKEVTL HKDGPLGETV LECYNCGCRN VFLLGFIPAK ADSVVVLLCR QPCASQSSLK DINWDSSQWQ PLIQDRCFLS WLVKIPSEQE QLRARQITAQ QINKLEELWK ENPSATLEDL EKPGVDEEPQ HVLLRYEDAY QYQNIFGPLV KLEADYDKKL KESQTQDNIT VRWDLGLNKK RIAYFTLPKT DSDMRLMQGD EICLRYKGDL APLWKGIGHV IKVPDNYGDE IAIELRSSVG APVEVTHNFQ VDFVWKSTSF DRMQSALKTF AVDETSVSGY IYHKLLGHEV EDVIIKCQLP KRFTAQGLPD LNHSQVYAVK TVLQRPLSLI QGPPGTGKTV TSATIVYHLA RQGNGPVLVC APSNIAVDQL TEKIHQTGLK VVRLCAKSRE AIDSPVSFLA LHNQIRNMDS MPELQKLQQL KDETGELSSA DEKRYRALKR TAERELLMNA DVICCTCVGA GDPRLAKMQF RSILIDESTQ ATEPECMVPV VLGAKQLILV GDHCQLGPVV MCKKAAKAGL SQSLFERLVV LGIRPIRLQV QYRMHPALSA FPSNIFYEGS LQNGVTAADR VKKGFDFQWP QPDKPMFFYV TQGQEEIASS GTSYLNRTEA ANVEKITTKL LKAGAKPDQI GIITPYEGQR SYLVQYMQFS GSLHTKLYQE VEIASVDAFQ GREKDFIILS CVRANEHQGI GFLNDPRRLN VALTRARYGV IIVGNPKALS KQPLWNHLLN YYKEQKVLVE GPLNNLRESL MQFSKPRKLV NTINPGARFM TTAMYDAREA IIPGSVYDRS SQGRPSSMYF QTHDQIGMIS AGPSHVAAMN IPIPFNLVMP PMPPPGYFGQ ANGPAAGRGT PKGKTGRGGR QKNRFGLPGP SQTNLPNSQA SQDVASQPFS QGALTQGYIS MSQPSQMSQP GLSQPELSQD SYLGDEFKSQ IDVALSQDST YQGERAYQHG GVTGLSQY

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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: 7.4
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 22000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 60.0 e/Å2

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.51 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 100475
FSC plot (resolution estimation)

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