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- EMDB-64959: Cryo-EM structure of SARS-CoV-2 nsp10/14:RNA-ATMP complex (focuse... -

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Basic information

Entry
Database: EMDB / ID: EMD-64959
TitleCryo-EM structure of SARS-CoV-2 nsp10/14:RNA-ATMP complex (focused on ExoN)
Map data
Sample
  • Complex: SARS-CoV-2 nsp10/14:RNA-ATMP
    • Protein or peptide: SARS-CoV-2 nsp10
    • Protein or peptide: SARS-CoV-2 nsp14
    • RNA: RNA (26-MER)
    • RNA: RNA (28-MER)
KeywordsSARS-CoV-2 / exonuclease / nsp14 / nucleotide analogue / VIRAL PROTEIN/RNA / VIRAL PROTEIN-RNA complex
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsWang J / Lou Z / Liu D
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32188101 China
National Natural Science Foundation of China (NSFC)22477122 China
CitationJournal: J Am Chem Soc / Year: 2025
Title: Structural Basis and Rational Design of Nucleotide Analogue Inhibitor Evading the SARS-CoV-2 Proofreading Enzyme.
Authors: Junbo Wang / Yufan Pan / Yixiao Liu / Bo Huang / Ge Jin / Lejin Zhang / Feng Zhou / Xiaoyu Chang / Yucen Huang / Liming Yan / Yuanchen Dong / Zihe Rao / Dongsheng Liu / Zhiyong Lou /
Abstract: All coronaviruses (CoVs) encode an exoribonuclease in nonstructural protein nsp14 (nsp14 ExoN), which is required for the excision of mismatched nucleotides or nucleotide analogues (NAs) that are ...All coronaviruses (CoVs) encode an exoribonuclease in nonstructural protein nsp14 (nsp14 ExoN), which is required for the excision of mismatched nucleotides or nucleotide analogues (NAs) that are incorporated into nascent RNA. Here, we investigated the mechanism by which NAs evade SARS-CoV-2 nsp14 ExoN cleavage using chemically synthesized RNA with NAs incorporated at the 3' end. Nsp14 ExoN exhibited significantly attenuated activity on RNA with sofosbuvir monophosphate (SMP) compared with natural nucleotides, remdesivir/molnupiravir monophosphate, and, in particular, AT-9010 monophosphate (ATMP), which has the same chemically modified ribose moiety as SMP, incorporated at the 3' end. Cryo-electron microscopy structures of nsp10/14 bound to RNA-SMP/-ATMP and mutagenesis studies revealed the essential roles of H95/Q145/F146 in recognizing the base moiety and thus pulling the NAs into a favored conformation for cleavage. Therefore, NAs may evade nsp14 ExoN cleavage by having (1) a base that does not interact with H95, Q145, or F146 and (2) a chemically modified ribose. Guided by this hypothesis, two NAs were designed to effectively resist nsp14 ExoN cleavage. These results inform the rational design of anti-CoV NAs.
History
DepositionJun 6, 2025-
Header (metadata) releaseJul 16, 2025-
Map releaseJul 16, 2025-
UpdateJul 16, 2025-
Current statusJul 16, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_64959.png

Downloads & links

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Map

FileDownload / File: emd_64959.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 400 pix.
= 292. Å		0.73 Å/pix.
x 400 pix.
= 292. Å		0.73 Å/pix.
x 400 pix.
= 292. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.73 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.8252769 - 2.669841
Average (Standard dev.)-0.00014205166 (±0.056390084)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 292.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_64959_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_64959_half_map_2.map
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Sample components

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Entire : SARS-CoV-2 nsp10/14:RNA-ATMP

EntireName: SARS-CoV-2 nsp10/14:RNA-ATMP
Components
  • Complex: SARS-CoV-2 nsp10/14:RNA-ATMP
    • Protein or peptide: SARS-CoV-2 nsp10
    • Protein or peptide: SARS-CoV-2 nsp14
    • RNA: RNA (26-MER)
    • RNA: RNA (28-MER)

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Supramolecule #1: SARS-CoV-2 nsp10/14:RNA-ATMP

SupramoleculeName: SARS-CoV-2 nsp10/14:RNA-ATMP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Macromolecule #1: SARS-CoV-2 nsp10

MacromoleculeName: SARS-CoV-2 nsp10 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AGNATEVPAN STVLSFCAFA VDAAKAYKDY LASGGQPITN CVKMLCTHTG TGQAITVTPE ANMDQESFGG ASCCLYCRCH IDHPNPKGFC DLKGKYVQIP TTCANDPVGF TLKNTVCTVC GMWKGYGCSC DQLREPMLQ

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Macromolecule #2: SARS-CoV-2 nsp14

MacromoleculeName: SARS-CoV-2 nsp14 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: ENVTGLFKDC SKVITGLHPT QAPTHLSVDT KFKTEGLCVD IPGIPKDMTY RRLISMMGFK MNYQVNGYPN MFITREEAIR HVRAWIGFDV EGCHATREAV GTNLPLQ LG FSTGVNLVAV PTGYVDTPNN TDFSRVSAKP PPGDQFKHLI PLMYKGLPWN VVRIKIVQML ...String:
ENVTGLFKDC SKVITGLHPT QAPTHLSVDT KFKTEGLCVD IPGIPKDMTY RRLISMMGFK MNYQVNGYPN MFITREEAIR HVRAWIGFDV EGCHATREAV GTNLPLQ LG FSTGVNLVAV PTGYVDTPNN TDFSRVSAKP PPGDQFKHLI PLMYKGLPWN VVRIKIVQML SDTLKNLSDR VVFVLWAHGF ELTSMKYFVK IGPERTCCLC DRRATCFSTA SDTYACWHHS IGFDYVYNPF MIDVQQWGFT GNLQSNHDLY CQVHGNAHVA SCDAIMTRCL AVHECFVKRV DWTIEYPIIG DELKINAACR KVQHMVVKAA LLADKFPVLH DIGNPKAIKC VPQADVEWKF YDAQPCSDKA YKIEELFYSY ATHSDKFTDG VCLFWNCNVD RYPANSIVCR FDTRVLSNLN LPGCDGGSLY VNKHAFHTP AFDKSAFVNL KQLPFFYYSD SPCESHGKQV VSDIDYVPLK SATCITRCNL GGAVCRHHAN EYRLYLDAYN MMISAGFSLW VYKQFDTYNL WNTFTRL

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Macromolecule #3: RNA (26-MER)

MacromoleculeName: RNA (26-MER) / type: rna / ID: 3
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
SequenceString:
UUCUCCUAAG AAGCUAUUAA AAUCACAT

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Macromolecule #4: RNA (28-MER)

MacromoleculeName: RNA (28-MER) / type: rna / ID: 4
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
SequenceString:
AAGUGAUUUU AAUAGCUUCU UAGGAUGA

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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: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.6 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: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 71299
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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