EMDB-38422: CryoEM structure of compound HNC-1664 bound with RdRP-RNA complex...

Basic information

Entry

Database: EMDB / ID: EMD-38422

• Title: CryoEM structure of compound HNC-1664 bound with RdRP-RNA complex of SARS-CoV-2
• Map data: Map of SARS-CoV-2 RNA dependent RNA Polymerase-RNA substrate-compound HNC-1664

Sample

• Complex: CryoEM structure of compound HNC-1644 bound with RdRP-RNA
  • Protein or peptide: RNA-directed RNA polymerase nsp12
  • Protein or peptide: Non-structural protein 8
  • Protein or peptide: Non-structural protein 7
  • RNA: RNA (5'-R(P*CP*UP*AP*CP*GP*CP*GP*UP*AP*GP*CP*AP*UP*G)-3')
  • RNA: RNA (5'-R(P*UP*UP*CP*AP*UP*GP*CP*UP*AP*CP*GP*CP*GP*UP*AP*G)-3')
• Ligand: [[(2~{R},3~{R},4~{S},5~{R})-4-fluoranyl-5-(5-iodanyl-4-methyl-pyrrolo[2,3-d]pyrimidin-7-yl)-3-oxidanyl-oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate
• Ligand: MAGNESIUM ION

• Keywords: RNA dependent RNA polymerase / SARS-CoV-2 / nucleoside analog / VIRAL PROTEIN / VIRAL PROTEIN-RNA complex

Function / homology

Function:

protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / ISG15-specific peptidase activity / TRAF3-dependent IRF activation pathway / Transcription of SARS-CoV-2 sgRNAs / Translation of Replicase and Assembly of the Replication Transcription Complex / snRNP Assembly / Replication of the SARS-CoV-2 genome / double membrane vesicle viral factory outer membrane / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / host cell endoplasmic reticulum-Golgi intermediate compartment / SARS coronavirus main proteinase / 3'-5'-RNA exonuclease activity / 5'-3' DNA helicase activity / host cell endosome / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / symbiont-mediated suppression of host toll-like receptor signaling pathway / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / omega peptidase activity / SARS-CoV-2 modulates host translation machinery / mRNA (guanine-N7)-methyltransferase / host cell Golgi apparatus / methyltransferase cap1 / symbiont-mediated perturbation of host ubiquitin-like protein modification / symbiont-mediated suppression of host NF-kappaB cascade / DNA helicase / methyltransferase cap1 activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / regulation of autophagy / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / host cell endoplasmic reticulum membrane / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated suppression of host gene expression / copper ion binding / viral translational frameshifting / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / lipid binding / host cell nucleus / SARS-CoV-2 activates/modulates innate and adaptive immune responses / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane

Domain/homology:

RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus Nsp12 Interface domain profile. / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / Nidovirus 2-O-methyltransferase / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Non-structural protein NSP15, middle domain superfamily / Coronavirus replicase NSP15, N-terminal oligomerization / Nonstructural protein 15, middle domain, coronavirus / Nonstructural protein 13, 1B domain, coronavirus / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Nidovirus 3'-5' exoribonuclease domain / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Non-structural protein NSP16, coronavirus-like / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Arterivirus Nsp11 N-terminal/Coronavirus NSP15 middle domain / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Endoribonuclease EndoU-like / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / : / Lipocalin signature. / DNA2/NAM7 helicase-like, C-terminal / AAA domain / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP3, N-terminal, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal / NSP1 globular domain superfamily, betacoronavirus / Non-structural protein 2, SARS-CoV-like / Carbamoyl-phosphate synthase subdomain signature 2. / Betacoronavirus Nsp3c-M domain profile. / NSP1, globular domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus replicase NSP1 / Betacoronavirus single-stranded poly(A) binding domain / NSP1, C-terminal domain, betacoronavirus / : / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / Betacoronavirus Nsp3e group 2-specific marker (G2M) domain profile. / Betacoronavirus Nsp3c-C domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Non-structural protein NSP3, nucleic acid-binding domain superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Papain-like viral protease, palm and finger domains, coronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / Coronavirus replicase NSP2, N-terminal / : / Coronavirus replicase NSP2, C-terminal / Coronavirus (CoV) Nsp2 middle domain profile. / NSP1, globular domain, alpha/betacoronavirus / Coronavirus (CoV) Nsp1 globular domain profile. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / Nonstructural protein 2, N-terminal domain, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / Peptidase family C16 domain profile. / : / : / Coronavirus replicase NSP7 / Coronavirus 3Ecto domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile.

Component:

Replicase polyprotein 1ab

• Biological species: Severe acute respiratory syndrome coronavirus / Severe acute respiratory syndrome coronavirus 2 / synthetic construct (others)
• Method: single particle reconstruction / cryo EM / Resolution: 3.29 Å
• Authors: Li M / An L / Hong Y / Li S / Zhang K / Gong Q / Chang C

Funding support

China, 1 items

Organization	Grant number	Country
Other government		China

• Citation: Journal: Nat Commun / Year: 2024; Title: An adenosine analog shows high antiviral potency against coronavirus and arenavirus mainly through an unusual base pairing mode.; Authors: Xiaoying Jia / Xuping Jing / Ming Li / Minli Gao / Yao Zhong / Entao Li / Yang Liu / Rui Li / Guoqiang Yao / Qiaojie Liu / Minmin Zhou / Yuxia Hou / Linfeng An / Yibao Hong / Shanshan Li / Jiancun Zhang / Wei Wang / Kaiming Zhang / Peng Gong / Sandra Chiu / ; Abstract: By targeting the essential viral RNA-dependent RNA polymerase (RdRP), nucleoside analogs (NAs) have exhibited great potential in antiviral therapy for RNA virus-related diseases. However, most ribose-modified NAs do not present broad-spectrum features, likely due to differences in ribose-RdRP interactions across virus families. Here, we show that HNC-1664, an adenosine analog with modifications both in ribose and base, has broad-spectrum antiviral activity against positive-strand coronaviruses and negative-strand arenaviruses. Importantly, treatment with HNC-1664 demonstrate anti-SARS-CoV-2 efficacy in infected K18-human ACE2 mice, with reduced viral titer and mortality, as well as improved lung injury. Enzymology data demonstrate that HNC-1664 inhibits RNA synthesis mainly at the pre-catalysis stage. The cryo-EM structures of HNC-1664-bound RdRP-RNA complexes from both SARS-CoV-2 and LASV reveal an unusual base pairing mode of HNC-1664 in part due to its base modification, thus revealing its great potency in binding but not catalysis. Under certain circumstances, 1664-TP can be slowly incorporated by RdRP through regular Watson-Crick base pairing, as evidenced by enzymology data and an HNC-1664-incorporated crystal structure of the RdRP-RNA complex. Overall, HNC-1664 achieves broad-spectrum characteristics by favoring an alternative base pairing strategy to non-catalytically block RNA synthesis, providing a novel concept for the rational development of NA drugs.

History

Deposition	Dec 23, 2023	-
Header (metadata) release	Dec 4, 2024	-
Map release	Dec 4, 2024	-
Update	Jan 15, 2025	-
Current status	Jan 15, 2025	Processing site: PDBj / Status: Released

Map

• File: Download / File: emd_38422.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Map of SARS-CoV-2 RNA dependent RNA Polymerase-RNA substrate-compound HNC-1664
• Voxel size: X=Y=Z: 0.82 Å

Density

Contour Level	By AUTHOR: 0.089
Minimum - Maximum	-0.9576972 - 1.430564
Average (Standard dev.)	-0.00005996195 (±0.03218066)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 336 336 336 Spacing 336 336 336
Cell	A=B=C: 275.52 Å α=β=γ: 90.0 °

Supplemental data

Additional map: Map of SARS-CoV-2 RNA dependent RNA Polymerase-RNA substrate-compound...

• File: emd_38422_additional_1.map
• Annotation: Map of SARS-CoV-2 RNA dependent RNA Polymerase-RNA substrate-compound HNC-1664

Half map: Half map of SARS-CoV-2 RNA dependent RNA Polymerase-RNA...

• File: emd_38422_half_map_1.map
• Annotation: Half map of SARS-CoV-2 RNA dependent RNA Polymerase-RNA substrate-compound HNC-1664

Half map: Half map of SARS-CoV-2 RNA dependent RNA Polymerase-RNA...

• File: emd_38422_half_map_2.map
• Annotation: Half map of SARS-CoV-2 RNA dependent RNA Polymerase-RNA substrate-compound HNC-1664

Sample components

Entire : CryoEM structure of compound HNC-1644 bound with RdRP-RNA

• Entire: Name: CryoEM structure of compound HNC-1644 bound with RdRP-RNA

Components

• Complex: CryoEM structure of compound HNC-1644 bound with RdRP-RNA
  • Protein or peptide: RNA-directed RNA polymerase nsp12
  • Protein or peptide: Non-structural protein 8
  • Protein or peptide: Non-structural protein 7
  • RNA: RNA (5'-R(P*CP*UP*AP*CP*GP*CP*GP*UP*AP*GP*CP*AP*UP*G)-3')
  • RNA: RNA (5'-R(P*UP*UP*CP*AP*UP*GP*CP*UP*AP*CP*GP*CP*GP*UP*AP*G)-3')
• Ligand: [[(2~{R},3~{R},4~{S},5~{R})-4-fluoranyl-5-(5-iodanyl-4-methyl-pyrrolo[2,3-d]pyrimidin-7-yl)-3-oxidanyl-oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate
• Ligand: MAGNESIUM ION

Supramolecule #1: CryoEM structure of compound HNC-1644 bound with RdRP-RNA

• Supramolecule: Name: CryoEM structure of compound HNC-1644 bound with RdRP-RNA; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
• Source (natural): Organism: Severe acute respiratory syndrome coronavirus
• Molecular weight: Theoretical: 150 KDa

Macromolecule #1: RNA-directed RNA polymerase nsp12

• Macromolecule: Name: RNA-directed RNA polymerase nsp12 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
• Source (natural): Organism: Severe acute respiratory syndrome coronavirus 2
• Molecular weight: Theoretical: 108.350703 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MGSADAQSFL NRVCGVSAAR LTPCGTGTST DVVYRAFDIY NDKVAGFAKF LKTNCCRFQE KDEDDNLIDS YFVVKRHTFS NYQHEETIY NLLKDCPAVA KHDFFKFRID GDMVPHISRQ RLTKYTMADL VYALRHFDEG NCDTLKEILV TYNCCDDDYF N KKDWYDFV ENPDILRVYA NLGERVRQAL LKTVQFCDAM RNAGIVGVLT LDNQDLNGNW YDFGDFIQTT PGSGVPVVDS YY SLLMPIL TLTRALTAES HVDTDLTKPY IKWDLLKYDF TEERLKLFDR YFKYWDQTYH PNCVNCLDDR CILHCANFNV LFS TVFPPT SFGPLVRKIF VDGVPFVVST GYHFRELGVV HNQDVNLHSS RLSFKELLVY AADPAMHAAS GNLLLDKRTT CFSV AALTN NVAFQTVKPG NFNKDFYDFA VSKGFFKEGS SVELKHFFFA QDGNAAISDY DYYRYNLPTM CDIRQLLFVV EVVDK YFDC YDGGCINANQ VIVNNLDKSA GFPFNKWGKA RLYYDSMSYE DQDALFAYTK RNVIPTITQM NLKYAISAKN RARTVA GVS ICSTMTNRQF HQKLLKSIAA TRGATVVIGT SKFYGGWHNM LKTVYSDVEN PHLMGWDYPK CDRAMPNMLR IMASLVL AR KHTTCCSLSH RFYRLANECA QVLSEMVMCG GSLYVKPGGT SSGDATTAYA NSVFNICQAV TANVNALLST DGNKIADK Y VRNLQHRLYE CLYRNRDVDT DFVNEFYAYL RKHFSMMILS DDAVVCFNST YASQGLVASI KNFKSVLYYQ NNVFMSEAK CWTETDLTKG PHEFCSQHTM LVKQGDDYVY LPYPDPSRIL GAGCFVDDIV KTDGTLMIER FVSLAIDAYP LTKHPNQEYA DVFHLYLQY IRKLHDELTG HMLDMYSVML TNDNTSRYWE PEFYEAMYTP HTVLQHHHHH HHHHH

UniProtKB: Replicase polyprotein 1ab

Macromolecule #2: Non-structural protein 8

• Macromolecule: Name: Non-structural protein 8 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Severe acute respiratory syndrome coronavirus
• Molecular weight: Theoretical: 23.455707 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

HHHHHHENLY FQGAIASEFS SLPSYAAFAT AQEAYEQAVA NGDSEVVLKK LKKSLNVAKS EFDRDAAMQR KLEKMADQAM TQMYKQARS EDKRAKVTSA MQTMLFTMLR KLDNDALNNI INNARDGCVP LNIIPLTTAA KLMVVIPDYN TYKNTCDGTT F TYASALWE IQQVVDADSK IVQLSEISMD NSPNLAWPLI VTALRANSAV KL

UniProtKB: Replicase polyprotein 1ab

Macromolecule #3: Non-structural protein 7

• Macromolecule: Name: Non-structural protein 7 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Severe acute respiratory syndrome coronavirus
• Molecular weight: Theoretical: 10.077685 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

SKMSDVKCTS VVLLSVLQQL RVESSSKLWA QCVQLHNDIL LAKDTTEAFE KMVSLLSVLL SMQGAVDINK LCEEMLDNRA TLQHHHHHH

UniProtKB: Replicase polyprotein 1ab

Macromolecule #4: RNA (5'-R(P*CP*UP*AP*CP*GP*CP*GP*UP*AP*GP*CP*AP*UP*G)-3')

• Macromolecule: Name: RNA (5'-R(P*CP*UP*AP*CP*GP*CP*GP*UP*AP*GP*CP*AP*UP*G)-3'); type: rna / ID: 4 / Number of copies: 1
• Source (natural): Organism: synthetic construct (others)
• Molecular weight: Theoretical: 4.462708 KDa

Sequence

String:

CUACGCGUAG CAUG

Macromolecule #5: RNA (5'-R(P*UP*UP*CP*AP*UP*GP*CP*UP*AP*CP*GP*CP*GP*UP*AP*G)-3')

• Macromolecule: Name: RNA (5'-R(P*UP*UP*CP*AP*UP*GP*CP*UP*AP*CP*GP*CP*GP*UP*AP*G)-3'); type: rna / ID: 5 / Number of copies: 1
• Source (natural): Organism: synthetic construct (others)
• Molecular weight: Theoretical: 5.993537 KDa

Sequence

String:

UUUUUCAUGC UACGCGUAG

Macromolecule #6: [[(2~{R},3~{R},4~{S},5~{R})-4-fluoranyl-5-(5-iodanyl-4-methyl-pyr...

• Macromolecule: Name: [[(2~{R},3~{R},4~{S},5~{R})-4-fluoranyl-5-(5-iodanyl-4-methyl-pyrrolo[2,3-d]pyrimidin-7-yl)-3-oxidanyl-oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate; type: ligand / ID: 6 / Number of copies: 1 / Formula: A1LVZ
• Molecular weight: Theoretical: 633.092 Da

Macromolecule #7: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 3 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 5.0 mg/mL
• Buffer: pH: 7.5
• Vitrification: Cryogen name: ETHANE

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.4 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.6 µm
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 1688273
• Startup model: Type of model: NONE
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2) / Number images used: 467095
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD
• Final angle assignment: Type: MAXIMUM LIKELIHOOD