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- EMDB-30284: COVID-19 RNA-dependent RNA polymerase pre-translocated catalytic ... -

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Basic information

Entry
Database: EMDB / ID: EMD-30284
TitleCOVID-19 RNA-dependent RNA polymerase pre-translocated catalytic complex in conformation II
Map data
Sample
  • Complex: COVID-19 RNA-dependent RNA polymerase pre-translocated catalytic complex in conformation II
    • Protein or peptide: RNA-directed RNA polymeraseRNA-dependent RNA polymerase
    • Protein or peptide: Non-structural protein 8
    • Protein or peptide: Non-structural protein 7
    • RNA: RNA (5'-R(P*GP*GP*GP*AP*GP*CP*UP*CP*CP*UP*CP*CP*UP*GP*UP*GP*UP*CP*GP*U)-3')
    • RNA: RNA (5'-R(P*AP*CP*GP*AP*CP*AP*CP*AP*GP*G*(GS4)P*G)-3')
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Foot-and-mouth disease virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsWang Q / Gao Y / Ji W / Mu A / Rao Z
Funding support China, 7 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2017YFC0840300 China
Ministry of Science and Technology (MoST, China)2018YFA0507200 China
Ministry of Science and Technology (MoST, China)2020YFA0707500 China
National Natural Science Foundation of China (NSFC)81520108019 China
Chinese Academy of SciencesXDB08020200 China
National Natural Science Foundation of China (NSFC)32041007 China
National Natural Science Foundation of China (NSFC)813300237 China
CitationJournal: Cell / Year: 2020
Title: Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.
Authors: Quan Wang / Jiqin Wu / Haofeng Wang / Yan Gao / Qiaojie Liu / An Mu / Wenxin Ji / Liming Yan / Yan Zhu / Chen Zhu / Xiang Fang / Xiaobao Yang / Yucen Huang / Hailong Gao / Fengjiang Liu / Ji ...Authors: Quan Wang / Jiqin Wu / Haofeng Wang / Yan Gao / Qiaojie Liu / An Mu / Wenxin Ji / Liming Yan / Yan Zhu / Chen Zhu / Xiang Fang / Xiaobao Yang / Yucen Huang / Hailong Gao / Fengjiang Liu / Ji Ge / Qianqian Sun / Xiuna Yang / Wenqing Xu / Zhijie Liu / Haitao Yang / Zhiyong Lou / Biao Jiang / Luke W Guddat / Peng Gong / Zihe Rao /
Abstract: Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete ...Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.
History
DepositionMay 10, 2020-
Header (metadata) releaseJun 3, 2020-
Map releaseJun 3, 2020-
UpdateAug 5, 2020-
Current statusAug 5, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30284.png

Downloads & links

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Map

FileDownload / File: emd_30284.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-1.0649309 - 2.2318783
Average (Standard dev.)0.0026022745 (±0.07152373)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 262.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z262.400262.400262.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ352352352
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-1.0652.2320.003

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Supplemental data

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Mask #1

Fileemd_30284_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_30284_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_30284_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : COVID-19 RNA-dependent RNA polymerase pre-translocated catalytic ...

EntireName: COVID-19 RNA-dependent RNA polymerase pre-translocated catalytic complex in conformation II
Components
  • Complex: COVID-19 RNA-dependent RNA polymerase pre-translocated catalytic complex in conformation II
    • Protein or peptide: RNA-directed RNA polymeraseRNA-dependent RNA polymerase
    • Protein or peptide: Non-structural protein 8
    • Protein or peptide: Non-structural protein 7
    • RNA: RNA (5'-R(P*GP*GP*GP*AP*GP*CP*UP*CP*CP*UP*CP*CP*UP*GP*UP*GP*UP*CP*GP*U)-3')
    • RNA: RNA (5'-R(P*AP*CP*GP*AP*CP*AP*CP*AP*GP*G*(GS4)P*G)-3')

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Supramolecule #1: COVID-19 RNA-dependent RNA polymerase pre-translocated catalytic ...

SupramoleculeName: COVID-19 RNA-dependent RNA polymerase pre-translocated catalytic complex in conformation II
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: full-length COVID-19 nsp12 (residues S1-Q932) was incubated with nsp7 (residues S1-Q83) and nsp8 (A1-Q198), and in complex with RNA template and product.
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: RNA-directed RNA polymerase

MacromoleculeName: RNA-directed RNA polymerase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSADAQSFL NRVCGVSAAR LTPCGTGTST DVVYRAFDIY NDKVAGFAKF LKTNCCRFQE KDEDDNLIDS YFVVKRHTFS NYQHEETIY NLLKDCPAVA KHDFFKFRID GDMVPHISRQ RLTKYTMADL VYALRHFDEG NCDTLKEILV TYNCCDDDYF N KKDWYDFV ...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

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Macromolecule #2: Non-structural protein 8

MacromoleculeName: Non-structural protein 8 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPAIASEFSS LPSYAAFATA QEAYEQAVAN GDSEVVLKKL KKSLNVAKSE FDRDAAMQRK LEKMADQAMT QMYKQARSED KRAKVTSAM QTMLFTMLRK LDNDALNNII NNARDGCVPL NIIPLTTAAK LMVVIPDYNT YKNTCDGTTF TYASALWEIQ Q VVDADSKI ...String:
GPAIASEFSS LPSYAAFATA QEAYEQAVAN GDSEVVLKKL KKSLNVAKSE FDRDAAMQRK LEKMADQAMT QMYKQARSED KRAKVTSAM QTMLFTMLRK LDNDALNNII NNARDGCVPL NIIPLTTAAK LMVVIPDYNT YKNTCDGTTF TYASALWEIQ Q VVDADSKI VQLSEISMDN SPNLAWPLIV TALRANSAVK LQ

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Macromolecule #3: Non-structural protein 7

MacromoleculeName: Non-structural protein 7 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GPSKMSDVKC TSVVLLSVLQ QLRVESSSKL WAQCVQLHND ILLAKDTTEA FEKMVSLLSV LLSMQGAVDI NKLCEEMLDN RATLQ

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

MacromoleculeName: RNA (5'-R(P*GP*GP*GP*AP*GP*CP*UP*CP*CP*UP*CP*CP*UP*GP*UP*GP*UP*CP*GP*U)-3')
type: rna / ID: 4
Source (natural)Organism: Foot-and-mouth disease virus
SequenceString:
GGGAGAUGUC UCCUCCUGUG UCGUCGAAA

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Macromolecule #5: RNA (5'-R(P*AP*CP*GP*AP*CP*AP*CP*AP*GP*G*(GS4)P*G)-3')

MacromoleculeName: RNA (5'-R(P*AP*CP*GP*AP*CP*AP*CP*AP*GP*G*(GS4)P*G)-3')
type: rna / ID: 5
Source (natural)Organism: Foot-and-mouth disease virus
SequenceString:
UGUUCGACGA CACAGG(GS4)G

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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

Concentration5 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K2 QUANTUM (4k x 4k) / #0 - Detector mode: SUPER-RESOLUTION / #0 - Average electron dose: 60.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 QUANTUM (4k x 4k) / #1 - Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 37696
Image recording ID1

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