EMDB-54695: Cryo-EM structure of SARS CoV-2 RdRp wild-type in complex with 20...

基本情報

登録情報

データベース: EMDB / ID: EMD-54695

• タイトル: Cryo-EM structure of SARS CoV-2 RdRp wild-type in complex with 20-40mer RNA incorporating ATP

試料

• 複合体: SARS CoV-2 RdRp wild-type in complex with 20-40mer RNA incorporating ATP
  • RNA: RNA primer strand (27-MER)
  • RNA: RNA template strand (40-MER)
  • タンパク質・ペプチド: RNA-directed RNA polymerase nsp12
  • タンパク質・ペプチド: Non-structural protein 8
  • タンパク質・ペプチド: Non-structural protein 7
• リガンド: ZINC ION

• キーワード: RNA-dependent RNA polymerase / SARS CoV-2 / transcription / viral protein / ATP / remdesivir / S759A mutant

機能・相同性

分子機能:

protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / 付加脱離酵素（リアーゼ）; P-Oリアーゼ類; - / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Maturation of replicase proteins / TRAF3-dependent IRF activation pathway / ISG15-specific peptidase activity / 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 / 加水分解酵素; エステル加水分解酵素; 5'-リン酸モノエステル産生エキソリボヌクレアーゼ / host cell endoplasmic reticulum-Golgi intermediate compartment / double membrane vesicle viral factory outer membrane / SARS coronavirus main proteinase / 5'-3' DNA helicase activity / 3'-5'-RNA exonuclease 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 / mRNA (guanine-N7)-methyltransferase / SARS-CoV-2 modulates host translation machinery / methyltransferase cap1 / host cell Golgi apparatus / symbiont-mediated suppression of host NF-kappaB cascade / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA helicase / methyltransferase cap1 activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / 加水分解酵素; プロテアーゼ; ペプチド結合加水分解酵素; システインプロテアーゼ / single-stranded RNA binding / host cell perinuclear region of cytoplasm / regulation of autophagy / 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 / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell nucleus / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane

ドメイン・相同性:

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.

構成要素:

Replicase polyprotein 1ab

• 生物種: Severe acute respiratory syndrome coronavirus 2 (ウイルス)
• 手法: 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 2.8 Å
• データ登録者: Oliva MF / Das K

資金援助

ベルギー, 1件

Organization	Grant number	国
KU Leuven		ベルギー

• 引用: ジャーナル: bioRxiv / 年: 2025; タイトル: Structural basis for selective remdesivir incorporation by SARS-CoV-2 RNA polymerase, and S759A resistance.; 著者: Calvin J Gordon / Mizar F Oliva / Hery W Lee / Quinten Goovaerts / Brent De Wijngaert / Matthias Götte / Kalyan Das; 要旨: Nucleoside analogs (NAs) have been successfully used to treat viral infections. dNTP analogs are primarily DNA chain terminators, while NTP analogs, such as remdesivir, can inhibit as delayed chain terminators or when in the template strand. Determining the frequency of remdesivir triphosphate (RTP) incorporation in the presence of the competing ATP can help in understanding different modes of viral RNA-dependent RNA polymerase (RdRp) inhibition by NTP analogs. We employed enzymatic assays, mass spectrometry, and cryo-EM to show that SARS-CoV-2 RdRp preferentially incorporates RTP, outcompeting 10-fold higher ATP concentration; however, successive RTP incorporations are less favoured when ATP is present. Structures of SARS-CoV-2 RdRp in this and previous studies demonstrate resilience of remdesivir:UMP base pair to translocation, explaining the reduced preference for conjugate incorporations. Together, the RTP versus ATP incorporation is driven by their relative concentration and structural rigidity of remdesivir:UMP, ultimately limiting the number of incorporated remdesivir in a fully synthesized RNA strand. The S759A mutant confers RTP resistance, and the structures of S759A RdRp catalytic complexes reveal that altered ribose-ring conformation and repositioning of the primer 3'-end nucleotide contribute to RTP resistance. These findings enhance our understanding of non-obligate NTP analogs and provide insight into S759A resistance mechanism.

履歴

登録	2025年8月7日	-
ヘッダ(付随情報) 公開	2025年10月1日	-
マップ公開	2025年10月1日	-
更新	2025年10月1日	-
現状	2025年10月1日	処理サイト: PDBe / 状態: 公開

マップ

• ファイル: ダウンロード / ファイル: emd_54695.map.gz / 形式: CCP4 / 大きさ: 15.6 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• ボクセルのサイズ: X=Y=Z: 0.9 Å

密度

表面レベル	登録者による: 0.0062
最小 - 最大	-0.04120943 - 0.07019061
平均 (標準偏差)	0.000049236274 (±0.0041623395)

• 対称性: 空間群: 1

詳細

EMDB XML:

マップ形状	Axis order X Y Z Origin 0 0 0 サイズ 160 160 160 Spacing 160 160 160
セル	A=B=C: 144.0 Å α=β=γ: 90.0 °

添付データ

マスク #1

• ファイル: emd_54695_msk_1.map

ハーフマップ: #2

• ファイル: emd_54695_half_map_1.map

ハーフマップ: #1

• ファイル: emd_54695_half_map_2.map

試料の構成要素

全体 : SARS CoV-2 RdRp wild-type in complex with 20-40mer RNA incorporat...

• 全体: 名称: SARS CoV-2 RdRp wild-type in complex with 20-40mer RNA incorporating ATP

要素

• 複合体: SARS CoV-2 RdRp wild-type in complex with 20-40mer RNA incorporating ATP
  • RNA: RNA primer strand (27-MER)
  • RNA: RNA template strand (40-MER)
  • タンパク質・ペプチド: RNA-directed RNA polymerase nsp12
  • タンパク質・ペプチド: Non-structural protein 8
  • タンパク質・ペプチド: Non-structural protein 7
• リガンド: ZINC ION

超分子 #1: SARS CoV-2 RdRp wild-type in complex with 20-40mer RNA incorporat...

• 超分子: 名称: SARS CoV-2 RdRp wild-type in complex with 20-40mer RNA incorporating ATP; タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#5
• 由来(天然): 生物種: Severe acute respiratory syndrome coronavirus 2 (ウイルス)

分子 #1: RNA primer strand (27-MER)

• 分子: 名称: RNA primer strand (27-MER) / タイプ: rna / ID: 1 / コピー数: 1
• 由来(天然): 生物種: Severe acute respiratory syndrome coronavirus 2 (ウイルス)
• 分子量: 理論値: 8.56412 KDa

配列

文字列:

GUCAUUCUCC UAAGAAGCUA UUAAAAU

分子 #2: RNA template strand (40-MER)

• 分子: 名称: RNA template strand (40-MER) / タイプ: rna / ID: 2 / コピー数: 1
• 由来(天然): 生物種: Severe acute respiratory syndrome coronavirus 2 (ウイルス)
• 分子量: 理論値: 12.720521 KDa

配列

文字列:

CUAUCCCCAU GUGAUUUUAA UAGCUUCUUA GGAGAAUGAC

分子 #3: RNA-directed RNA polymerase nsp12

• 分子: 名称: RNA-directed RNA polymerase nsp12 / タイプ: protein_or_peptide / ID: 3 / コピー数: 1 / 光学異性体: LEVO / EC番号: RNA-directed RNA polymerase
• 由来(天然): 生物種: Severe acute respiratory syndrome coronavirus 2 (ウイルス)
• 分子量: 理論値: 106.436648 KDa
• 組換発現: 生物種: Escherichia coli BL21(DE3) (大腸菌)

配列

文字列:

QSFLNRVCGV SAARLTPCGT GTSTDVVYRA FDIYNDKVAG FAKFLKTNCC RFQEKDEDDN LIDSYFVVKR HTFSNYQHEE TIYNLLKDC PAVAKHDFFK FRIDGDMVPH ISRQRLTKYT MADLVYALRH FDEGNCDTLK EILVTYNCCD DDYFNKKDWY D FVENPDIL RVYANLGERV RQALLKTVQF CDAMRNAGIV GVLTLDNQDL NGNWYDFGDF IQTTPGSGVP VVDSYYSLLM PI LTLTRAL TAESHVDTDL TKPYIKWDLL KYDFTEERLK LFDRYFKYWD QTYHPNCVNC LDDRCILHCA NFNVLFSTVF PPT SFGPLV RKIFVDGVPF VVSTGYHFRE LGVVHNQDVN LHSSRLSFKE LLVYAADPAM HAASGNLLLD KRTTCFSVAA LTNN VAFQT VKPGNFNKDF YDFAVSKGFF KEGSSVELKH FFFAQDGNAA ISDYDYYRYN LPTMCDIRQL LFVVEVVDKY FDCYD GGCI NANQVIVNNL DKSAGFPFNK WGKARLYYDS MSYEDQDALF AYTKRNVIPT ITQMNLKYAI SAKNRARTVA GVSICS TMT NRQFHQKLLK SIAATRGATV VIGTSKFYGG WHNMLKTVYS DVENPHLMGW DYPKCDRAMP NMLRIMASLV LARKHTT CC SLSHRFYRLA NECAQVLSEM VMCGGSLYVK PGGTSSGDAT TAYANSVFNI CQAVTANVNA LLSTDGNKIA DKYVRNLQ H RLYECLYRNR DVDTDFVNEF YAYLRKHFSM MILSDDAVVC FNSTYASQGL VASIKNFKSV LYYQNNVFMS EAKCWTETD LTKGPHEFCS QHTMLVKQGD DYVYLPYPDP SRILGAGCFV DDIVKTDGTL MIERFVSLAI DAYPLTKHPN QEYADVFHLY LQYIRKLHD ELTGHMLDMY SVMLTNDNTS RYWEPEFYEA MYTPHTVLQ

UniProtKB: Replicase polyprotein 1ab

分子 #4: Non-structural protein 8

• 分子: 名称: Non-structural protein 8 / タイプ: protein_or_peptide / ID: 4 / コピー数: 1 / 光学異性体: LEVO / EC番号: ubiquitinyl hydrolase 1
• 由来(天然): 生物種: Severe acute respiratory syndrome coronavirus 2 (ウイルス)
• 分子量: 理論値: 21.903047 KDa
• 組換発現: 生物種: Escherichia coli BL21(DE3) (大腸菌)

配列

文字列:

AIASEFSSLP SYAAFATAQE AYEQAVANGD SEVVLKKLKK SLNVAKSEFD RDAAMQRKLE KMADQAMTQM YKQARSEDKR AKVTSAMQT MLFTMLRKLD NDALNNIINN ARDGCVPLNI IPLTTAAKLM VVIPDYNTYK NTCDGTTFTY ASALWEIQQV V DADSKIVQ LSEISMDNSP NLAWPLIVTA LRANSAVKLQ

UniProtKB: Replicase polyprotein 1ab

分子 #5: Non-structural protein 7

• 分子: 名称: Non-structural protein 7 / タイプ: protein_or_peptide / ID: 5 / コピー数: 1 / 光学異性体: LEVO / EC番号: ubiquitinyl hydrolase 1
• 由来(天然): 生物種: Severe acute respiratory syndrome coronavirus 2 (ウイルス)
• 分子量: 理論値: 9.248804 KDa
• 組換発現: 生物種: Escherichia coli BL21(DE3) (大腸菌)

配列

文字列:

SKMSDVKCTS VVLLSVLQQL RVESSSKLWA QCVQLHNDIL LAKDTTEAFE KMVSLLSVLL SMQGAVDINK LCEEMLDNRA TLQ

UniProtKB: Replicase polyprotein 1ab

分子 #6: ZINC ION

• 分子: 名称: ZINC ION / タイプ: ligand / ID: 6 / コピー数: 2 / 式: ZN
• 分子量: 理論値: 65.409 Da

実験情報

構造解析

• 手法: クライオ電子顕微鏡法
• 解析: 単粒子再構成法
• 試料の集合状態: particle

試料調製

• 緩衝液: pH: 7.5 ; 詳細: 20 mM HEPES pH 7.5, 75 mM NaCl, 5 mM MgCl2, 5 mM DTT
• グリッド: モデル: Quantifoil R1.2/1.3 / 材質: GOLD / メッシュ: 300 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: HOLEY / 前処理 - タイプ: GLOW DISCHARGE
• 凍結: 凍結剤: ETHANE / チャンバー内湿度: 95 % / チャンバー内温度: 277.15 K / 装置: LEICA EM GP

電子顕微鏡法

• 顕微鏡: TFS GLACIOS
• 特殊光学系: エネルギーフィルター - 名称: TFS Selectris
• ソフトウェア: 名称: EPU
• 撮影: フィルム・検出器のモデル: FEI FALCON IV (4k x 4k); 撮影したグリッド数: 1 / 平均電子線量: 40.0 e/Ų
• 電子線: 加速電圧: 200 kV / 電子線源: FIELD EMISSION GUN
• 電子光学系: 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 最大 デフォーカス（公称値）: 1.8 µm / 最小 デフォーカス（公称値）: 0.6 µm / 倍率（公称値）: 130000
• 試料ステージ: 試料ホルダーモデル: OTHER / ホルダー冷却材: NITROGEN

画像解析

• CTF補正: タイプ: NONE

初期モデル

モデルのタイプ: PDB ENTRY
PDBモデル - PDB ID:

7dok

• 最終 再構成: 解像度のタイプ: BY AUTHOR / 解像度: 2.8 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: RELION / 使用した粒子像数: 109207
• 初期 角度割当: タイプ: MAXIMUM LIKELIHOOD
• 最終 角度割当: タイプ: MAXIMUM LIKELIHOOD