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- EMDB-18120: The closed state of the ASFV apo-RNA polymerase -

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Basic information

Entry
Database: EMDB / ID: EMD-18120
TitleThe closed state of the ASFV apo-RNA polymerase
Map dataSharpened map
Sample
  • Complex: apo-form of the 8-subunit RNA polymerase from African Swine Fever Virus
    • Protein or peptide: DNA-directed RNA polymerase RPB1 homolog
    • Protein or peptide: DNA-directed RNA polymerase RPB2 homolog
    • Protein or peptide: DNA-directed RNA polymerase RPB3-11 homolog
    • Protein or peptide: DNA-directed RNA polymerase RPB7 homolog
    • Protein or peptide: DNA-directed RNA polymerase RPB5 homolog
    • Protein or peptide: DNA-directed RNA polymerase RPB6 homolog
    • Protein or peptide: Uncharacterized protein C122R
    • Protein or peptide: DNA-directed RNA polymerase RPB10 homolog
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsRNA polymerase / ASFV / transcription / eukaryotic virus
Function / homology
Function and homology information


DNA-templated viral transcription / RNA polymerase III activity / viral transcription / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / DNA-directed RNA polymerase complex / virion component / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity ...DNA-templated viral transcription / RNA polymerase III activity / viral transcription / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / DNA-directed RNA polymerase complex / virion component / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / host cell cytoplasm / protein dimerization activity / DNA-templated transcription / DNA binding / zinc ion binding / metal ion binding
Similarity search - Function
RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. ...RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6
Similarity search - Domain/homology
DNA-directed RNA polymerase RPB6 homolog / DNA-directed RNA polymerase RPB1 homolog / DNA-directed RNA polymerase RPB2 homolog / DNA-directed RNA polymerase RPB10 homolog / Uncharacterized protein C122R / DNA-directed RNA polymerase RPB5 homolog / DNA-directed RNA polymerase RPB3-11 homolog / DNA-directed RNA polymerase RPB7 homolog
Similarity search - Component
Biological speciesAfrican swine fever virus BA71V
Methodsingle particle reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsPilotto S / Sykora M / Cackett G / Werner F
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/X017028/1 United Kingdom
Wellcome TrustWT207446/Z/17/Z United Kingdom
Wellcome TrustWT108877/B/15/Z United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Structure of the recombinant RNA polymerase from African Swine Fever Virus.
Authors: Simona Pilotto / Michal Sýkora / Gwenny Cackett / Christopher Dulson / Finn Werner /
Abstract: African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of ...African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of the infected cell and encodes its own transcription machinery that is independent of cellular factors, however, not much is known about how this system works at a molecular level. Here, we present methods to produce recombinant ASFV RNA polymerase, functional assays to screen for inhibitors, and high-resolution cryo-electron microscopy structures of the ASFV RNAP in different conformational states. The ASFV RNAP bears a striking resemblance to RNAPII with bona fide homologues of nine of its twelve subunits. Key differences include the fusion of the ASFV assembly platform subunits RPB3 and RPB11, and an unusual C-terminal domain of the stalk subunit vRPB7 that is related to the eukaryotic mRNA cap 2´-O-methyltransferase 1. Despite the high degree of structural conservation with cellular RNA polymerases, the ASFV RNAP is resistant to the inhibitors rifampicin and alpha-amanitin. The cryo-EM structures and fully recombinant RNAP system together provide an important tool for the design, development, and screening of antiviral drugs in a low biosafety containment environment.
History
DepositionAug 3, 2023-
Header (metadata) releaseFeb 14, 2024-
Map releaseFeb 14, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18120.png

Downloads & links

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Map

FileDownload / File: emd_18120.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.83 Å/pix.
x 300 pix.
= 248.4 Å		0.83 Å/pix.
x 300 pix.
= 248.4 Å		0.83 Å/pix.
x 300 pix.
= 248.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.5
Minimum - Maximum-15.950768 - 27.741410999999999
Average (Standard dev.)0.000000000001189 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 248.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened 3DFlex refinement map

Fileemd_18120_additional_1.map
Annotationunsharpened 3DFlex refinement map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3DFlex refinement half-map-A

Fileemd_18120_half_map_1.map
Annotation3DFlex refinement half-map-A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3DFlex refinement half-map-B

Fileemd_18120_half_map_2.map
Annotation3DFlex refinement half-map-B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : apo-form of the 8-subunit RNA polymerase from African Swine Fever...

EntireName: apo-form of the 8-subunit RNA polymerase from African Swine Fever Virus
Components
  • Complex: apo-form of the 8-subunit RNA polymerase from African Swine Fever Virus
    • Protein or peptide: DNA-directed RNA polymerase RPB1 homolog
    • Protein or peptide: DNA-directed RNA polymerase RPB2 homolog
    • Protein or peptide: DNA-directed RNA polymerase RPB3-11 homolog
    • Protein or peptide: DNA-directed RNA polymerase RPB7 homolog
    • Protein or peptide: DNA-directed RNA polymerase RPB5 homolog
    • Protein or peptide: DNA-directed RNA polymerase RPB6 homolog
    • Protein or peptide: Uncharacterized protein C122R
    • Protein or peptide: DNA-directed RNA polymerase RPB10 homolog
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: apo-form of the 8-subunit RNA polymerase from African Swine Fever...

SupramoleculeName: apo-form of the 8-subunit RNA polymerase from African Swine Fever Virus
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 450 KDa

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Macromolecule #1: DNA-directed RNA polymerase RPB1 homolog

MacromoleculeName: DNA-directed RNA polymerase RPB1 homolog / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 163.936094 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MEAGYAEIAA VQFNIAGDND HKRQGVMEVT ISNLFEGTLP AEGGIYDARM GTTDHHYKCI TCSHQRKQCM GHPGILQMHA PVLQPLFIA EIRRWLRVIC LNCGAPIVDL KRYEHLIRPK RLIEAASSQT EGKQCYVCKA VHPKIVKDSE DYFTFWVDQQ G KIDKLYPQ ...String:
MEAGYAEIAA VQFNIAGDND HKRQGVMEVT ISNLFEGTLP AEGGIYDARM GTTDHHYKCI TCSHQRKQCM GHPGILQMHA PVLQPLFIA EIRRWLRVIC LNCGAPIVDL KRYEHLIRPK RLIEAASSQT EGKQCYVCKA VHPKIVKDSE DYFTFWVDQQ G KIDKLYPQ IIREIFSRVT YDTVVKLGRS KNSHPEKLVL KAIQIPPISI RPGIRLGIGS GPQSFHDINN VIQYLVRKNL LI PKDLQIV RGQKIPLNID RNLQTIQQLY YNFLLDSVST TATQGGTGKR GIVMGARPAP SIMRRLPRKE GRIRKSLLGS QVW SISRST ICGNSDLHLD EVGYPISFAR TLQVAETVQH YNINRLMPYF LNGKRQYPGC SRVYKQITQS VHDIEGLKQD FRLE VGDIL YRDVVTGDVA FFNRQPSLER SSIGVHRIVV LENPKISTFQ MNVSACAWYN ADFDGDQMNL WVPWSVMSRV EAELL CSVR NWFISTKSSG PVNGQVQDST VGSFLLTRTN TPMGKNVMNK LHAMGLFQTT QTDPPCFANY SPTDLLDGKS VVSMLL KQT PINYQRAPTW YSEVYAPYMH YNKQDISTQI RNGELIEGVL DKKAVGAGSS GGIYHLISRR YGPQQALKMI FATQQLA LN YVRNAGFTVS TADMLLTPEA HQEVQEIINK LLLESEEINN RLLHGDIMPP IGLTTHDFYE KLQLNALKFP DRILKPIM N SINPETNGLF QMVATGAKGS NPNMIHIMAG IGQIEINTQR IQPQFSFGRT LVYYPRFALE AQAYGFICNS YIAGLTSPE FIFGEMNGRF DLINKALSTS STGYANRKAI FGLQSCIVDY YRRVSIDTRL VQQLYGEDGL DARQLETVRF ETIMLSDQEL EDKFKYTGI QSPLFEEEFS RLKKDRDKYR QIFLNVENFN FSQLLTDVRQ VPVNVASIVK NILLSSTSGV LPFDEKSILQ K YAMVKTFC KNLPYVFINN IQERLQTPIP VYLKRAAALM RMLIRIELAT VKTLNITCEQ MSAILDLIRL QYTQSLINYG EA VGILAAQ SVSEPLTQYM LDSHHRSVAG GTNKSGIVRP QEIFSAKPVE AEQSSEMLLR LKNPEVETNK TYAQEIANSI ELI TFERLI LQWHLLYETY SSTKKNVMYP DFASDVEWMT DFLENHPLLQ PPEDIANWCI RLELNKTTMI LKSISLESII NSLR AKHPN TYIMHSVENT ASGIPIIIRI YLRESAFRRS TNTRMATDEK IAVNVVDKLL NSTIRGIPGI KNANVVKLMR HRVDA QGKL VRLDNIYAIK TNGTNIFGAM LDDNIDPYTI VSSSIGDTME LYGIEAARQK IISEIRTVMG DKGPNHRHLL MYADLM TRT GQVTSLEKAG LNAREPSNVL LRMALSSPVQ VLTDAAVDSA VNPIYGIAAP TLMGSVPRIG TMYSDIIMDE KYITENY KS VDSMIDML

UniProtKB: DNA-directed RNA polymerase RPB1 homolog

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Macromolecule #2: DNA-directed RNA polymerase RPB2 homolog

MacromoleculeName: DNA-directed RNA polymerase RPB2 homolog / type: protein_or_peptide / ID: 2
Details: The protein was tagged at the N-terminus with a His-ZZ cleavable tag. The tag was cleaved with TEV protease leaving a G as the first residue
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 140.119406 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GMEPLRPQIT YGPIETVDNE ELTEADMLSF ISAAVNSTGL IGYNIKSFDD LMDNGIPQIV KQMFNVDITY KDQRDHTEID KLRESVQIQ FNFTDVNIER PQHRNYSQGN KINLLPNKAR LCGLSYSGPV NLAAEVILTA HYSNGRQEVK RASIPPFQVS T FPIMRGSN ...String:
GMEPLRPQIT YGPIETVDNE ELTEADMLSF ISAAVNSTGL IGYNIKSFDD LMDNGIPQIV KQMFNVDITY KDQRDHTEID KLRESVQIQ FNFTDVNIER PQHRNYSQGN KINLLPNKAR LCGLSYSGPV NLAAEVILTA HYSNGRQEVK RASIPPFQVS T FPIMRGSN RCHTHHLSKT AKKEIGEDPN EPGGYFIARG GEWVVDLLEN IRFNTLHIHY HTMQQGNNEI IRGEFISQPG GA FENSSQI IIRYMTTGAI TIEINSTKFS KLRIPWYLIF RMFGMTGDDS IIEQVVFDLE SNSPVNTFMI EILEKSIHVL DPI FQPVQH EPNREKIIQF LSEKVSKFVS NPSAYKSDEN AVQYLNERQL TILDKILLPH MGQTADTRVR KLRFLGLLIH KILL VIMNV FPPTDRDSYR TKRVHGSGVS LAKAFKAIFN TSVIAPIING FKELLKQTAF EELTQRNIIE AFSAALSKNT ASDLN RSME QSIISGNKTI MVRQRPIVNR VSTQSLERKN LLNTISALRT VNTHNTTNAS KQTERADMMR RVHASYPGYI CVAQSA DTG EKVGMSKQLA ITANVCTAGE VLSLKQRLLS DPAIQQLADV SNKDIVRKGL ARVFINGEWI GCCTNAFELA QRYRMFR RE GKIVHPHTTI YWDSMVDEVE FWLDVGRLTR PLLIVDNNIE KYNQACYKAA EARKKGDKDW EKHKIPFIQN TRFTSQMA K DILAGTLTLE DLVAQGICEF ITPEEAENCL VAFSIIELRK HKHDVTRRFT HVDVPQAILG LAALVSPYAN CTQPARVTY ETNQGRQTGG WYCFSWPYRV DMNRFFQFYN EMPLVKTIAH NYVIPNGLNT IVAYMIYGGY NQEDSVIVSQ SFIDRGGFAG TFYREEKVE LESDIESFGK PDPLITKNLK PGANYEKLVD GFVPVGTVVK KGDIIIGKVA KIRGEKDELN KYIDRSVMYG F DEPAVVDA VMRPHGPNDE IFGLMRLRYE RNLNIGDKMS SRSGNKGIAA LALPTSDMPF TEDGLQPDLI VNPHSHPSRM TN GQMIETT VGLANALQGV VTDGTAFLPI NVQLLSERLA QEGLRFNGCQ KMFNGQTGEY FDAAIFIGPT YHQRLQKFVL DDR YAVASY GPTDALTGQP LDGKRSHGGL RLGEMEHWVL TAQGAMQTII EKSHDDSDGC ISYICRNCGE PAIYNASHPI YKCM NCDVQ ADIGMVDSRR SSIVFQHEMR AANVNITSVL SPRVFQPA

UniProtKB: DNA-directed RNA polymerase RPB2 homolog

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Macromolecule #3: DNA-directed RNA polymerase RPB3-11 homolog

MacromoleculeName: DNA-directed RNA polymerase RPB3-11 homolog / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 41.352035 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MEKIFQNVEI KPFLIDFSNP FIKNAAKRLF QLEEQLPLVP VNVVMDFKGI SRAAVHGLSR VLQDEIPNYM LDIKPGGYKI EDSTDLFMT EQFIRNRINF IPIYAKNETL VFALRSLNNS CEVKTIYSRD LIQVAGPKLK YPIFNPTFEI GFLQPGKSLI I EDIYIKKG ...String:
MEKIFQNVEI KPFLIDFSNP FIKNAAKRLF QLEEQLPLVP VNVVMDFKGI SRAAVHGLSR VLQDEIPNYM LDIKPGGYKI EDSTDLFMT EQFIRNRINF IPIYAKNETL VFALRSLNNS CEVKTIYSRD LIQVAGPKLK YPIFNPTFEI GFLQPGKSLI I EDIYIKKG IGRKHAAFNL AVKTHFSHLD IEQYPTDKKE YMALSGYKQS SMTSDPRHHR LGLCFPAVPL PHINQAVRTY LK NACRIII GRIQSIQKIY ENFEEPQPEL VLFSLDEEKT KAIITIKDET HTIGNLLKTC IYEMIPDISF VGYQCVPHKQ EMV LTIIHK ASQEDLITLL EKSIQNIIQT FQILEKNVDE LIA

UniProtKB: DNA-directed RNA polymerase RPB3-11 homolog

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Macromolecule #4: DNA-directed RNA polymerase RPB7 homolog

MacromoleculeName: DNA-directed RNA polymerase RPB7 homolog / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 38.789609 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MIDQKIFETT LNIDDPTNFC TNVEAHLLKE LENIYVGKCF KNSFILNITG VIQRSPCFIM RTNNSGRGYM HVRFSAVVSY LNAFDLIAA VKIIKNDSNI ILGESLLTEP VTIVIPSSES QNNVAEVGQI VPVQLANSSV YYIPGRQQAS ATGSIFIPKH T FSVYHVQE ...String:
MIDQKIFETT LNIDDPTNFC TNVEAHLLKE LENIYVGKCF KNSFILNITG VIQRSPCFIM RTNNSGRGYM HVRFSAVVSY LNAFDLIAA VKIIKNDSNI ILGESLLTEP VTIVIPSSES QNNVAEVGQI VPVQLANSSV YYIPGRQQAS ATGSIFIPKH T FSVYHVQE ELTQEQALNL TKLVNIIEML LESRSKKDFK QICFFEKLYY TYSISSDEIL DLKIWKGPKG KEMSRLKPCN VL SFLYDAL KNKSSSLGFW ARPPNLLKSS PLAYQQDQNS FNATELPIIC SAEVMFVTLL KEIINYLQFM NDLCDTFNNE QLI KRHENI WMLIEQRKIG HDF

UniProtKB: DNA-directed RNA polymerase RPB7 homolog

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Macromolecule #5: DNA-directed RNA polymerase RPB5 homolog

MacromoleculeName: DNA-directed RNA polymerase RPB5 homolog / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 23.693711 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MAMQKLFTYI YEFIEYRKMV LLEEKVPYDK FVQMVLNTGF FRINAETLNH GIVSVFIFGA NGKYVHHGGD MRTLLTNTLN EKKHYEELI LIVDKPVLSK KNILDIIVEQ RAANPTIVIN IYPYHLFCIN IPKVSAIPKH KLITQEEAQE FLGREYLQPQ D LMQISASD ...String:
MAMQKLFTYI YEFIEYRKMV LLEEKVPYDK FVQMVLNTGF FRINAETLNH GIVSVFIFGA NGKYVHHGGD MRTLLTNTLN EKKHYEELI LIVDKPVLSK KNILDIIVEQ RAANPTIVIN IYPYHLFCIN IPKVSAIPKH KLITQEEAQE FLGREYLQPQ D LMQISASD PPVVWLGGRP GDFVQIERPS ETAMHAVVIR FITKSKI

UniProtKB: DNA-directed RNA polymerase RPB5 homolog

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Macromolecule #6: DNA-directed RNA polymerase RPB6 homolog

MacromoleculeName: DNA-directed RNA polymerase RPB6 homolog / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 16.704811 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MADNDNEDII MDDLVEEYVE TEEENFVDSE EESEDKDEIV ESPSICEGFV QASSQTLVII PDNERITSNV LTTFEATRLV AVRAQQLAI NGSTMLKKKY SSPIDIAKQE LFNRKIPLLV MRCIKVTPEG QKIVEIWNPR EMGIPLLD

UniProtKB: DNA-directed RNA polymerase RPB6 homolog

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Macromolecule #7: Uncharacterized protein C122R

MacromoleculeName: Uncharacterized protein C122R / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 11.831861 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MKICKACSSC MVRTYVDGNI IFRCSCGESV QGDSQNLLVS SKVYHTGEME DKYKIFIKNA PFDPTNCQIK KDCPNCHLDY LTQICIGSQ KIIILVCRCG YMSNRG

UniProtKB: Uncharacterized protein C122R

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Macromolecule #8: DNA-directed RNA polymerase RPB10 homolog

MacromoleculeName: DNA-directed RNA polymerase RPB10 homolog / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 9.098826 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MLIPVVCFTC GFPIGTYAAI FDKARTEYIK TKMDGTLPQN IPLDASLQIE LKDLITALGI PMRVCCRTHL ITTLDYRKYY

UniProtKB: DNA-directed RNA polymerase RPB10 homolog

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Macromolecule #9: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 9 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.05 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3tris
250.0 mMNaClsodium chloride
1.0 mMMgCl2magnesium chloride
0.1 mMZnSO4zinc sulfate
1.0 mMC4H10O2S2DTT
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
Details: The grid was covered with freshly prepared graphene oxide prior to use
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisparse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 14638 / Average exposure time: 2.6 sec. / Average electron dose: 48.152 e/Å2
Details: Images were collected in movie-mode for a total of 50 frames per image. The data collection was carried out in super-resolution mode and binned 2 on-the-fly.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsImages were motion corrected in Relion v4
Particle selectionNumber selected: 4842857
Startup modelType of model: NONE / Details: Initial model in cryoSPARC v3 and relion v4
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Software - details: 3D flexibility refinement / Number images used: 467000
Initial angle assignmentType: NOT APPLICABLE
Software:
Namedetails
cryoSPARC (ver. 3)2/3D classifications and 3DVA
RELION (ver. 4)3D refinement and post-processing

Details: Both relion and cryoSPARC were used
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 4) / Software - details: 3D flexibility refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 113 / Target criteria: cross-correlation coefficient
Output model

PDB-8q3b:
The closed state of the ASFV apo-RNA polymerase

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