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- EMDB-64958: Cryo-EM structure of SARS-CoV-2 nsp10/nsp14:RNA:SMP complex (focu... -

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Basic information

Entry
Database: EMDB / ID: EMD-64958
TitleCryo-EM structure of SARS-CoV-2 nsp10/nsp14:RNA:SMP complex (focused on ExoN)
Map data
Sample
  • Complex: SARS-CoV-2 nsp10/14:RNA-SMP
    • Protein or peptide: SARS-CoV-2 nsp10
    • Protein or peptide: SARS-CoV-2 nsp14
    • RNA: RNA (27-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: 3.79 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) releaseAug 20, 2025-
Map releaseAug 20, 2025-
UpdateAug 20, 2025-
Current statusAug 20, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_64958.png

Downloads & links

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Map

FileDownload / File: emd_64958.map.gz / Format: CCP4 / Size: 160.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 348 pix.
= 288.84 Å		0.83 Å/pix.
x 348 pix.
= 288.84 Å		0.83 Å/pix.
x 348 pix.
= 288.84 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.09056412 - 20.822303999999999
Average (Standard dev.)-0.03244625 (±0.27202818)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions348348348
Spacing348348348
CellA=B=C: 288.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_64958_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_64958_half_map_2.map
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Sample components

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

EntireName: SARS-CoV-2 nsp10/14:RNA-SMP
Components
  • Complex: SARS-CoV-2 nsp10/14:RNA-SMP
    • Protein or peptide: SARS-CoV-2 nsp10
    • Protein or peptide: SARS-CoV-2 nsp14
    • RNA: RNA (27-MER)
    • RNA: RNA (28-MER)

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

SupramoleculeName: SARS-CoV-2 nsp10/14:RNA-SMP / 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: AENVTGLFKD CSKVITGLHP TQAPTHLSVD TKFKTEGLCV DIPGIPKDMT YRRLISMMGF KMNYQVNGYP NMFITREEAI RHVRAWIGFD VEGCHATREA VGTNLPLQ L GFSTGVNLVA VPTGYVDTPN NTDFSRVSAK PPPGDQFKHL IPLMYKGLPW NVVRIKIVQM ...String:
AENVTGLFKD CSKVITGLHP TQAPTHLSVD TKFKTEGLCV DIPGIPKDMT YRRLISMMGF KMNYQVNGYP NMFITREEAI RHVRAWIGFD VEGCHATREA VGTNLPLQ L GFSTGVNLVA VPTGYVDTPN NTDFSRVSAK PPPGDQFKHL IPLMYKGLPW NVVRIKIVQM LSDTLKNLSD RVVFVLWAHG FELTSMKYFV KIGPERTCCL CDRRATCFST ASDTYACWHH SIGFDYVYNP FMIDVQQWGF TGNLQSNHDL YCQVHGNAHV ASCDAIMTRC LAVHECFVKR VDWTIEYPII GDELKINAAC RKVQHMVVKA ALLADKFPVL HDIGNPKAIK CVPQADVEWK FYDAQPCSDK AYKIEELFYS YATHSDKFTD GVCLFWNCNV DRYPANSIVC RFDTRVLSNL NLPGCDGGSL YVNKHAFHTP AFDKSAFVN LKQLPFFYYS DSPCESHGKQ VVSDIDYVPL KSATCITRCN LGGAVCRHHA NEYRLYLDAY NMMISAGFSL WVYKQFDTYN LWNTFTRL

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Macromolecule #3: RNA (27-MER)

MacromoleculeName: RNA (27-MER) / type: rna / ID: 3
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
SequenceString:
UUCUCCUAAG AAGCUAUUAA AAUCAC(6GS)

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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: GATAN K3 BIOQUANTUM (6k 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: 1.2 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: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: CRYO / Number images used: 179937
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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