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- EMDB-13026: Cryo-EM structure of the ATV RNAP Inhibitory Protein (RIP) bound ... -

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Basic information

Entry
Database: EMDB / ID: EMD-13026
TitleCryo-EM structure of the ATV RNAP Inhibitory Protein (RIP) bound to the DNA-binding channel of the host's RNA polymerase
Map dataPost-process map obtained from Relion 3.0
Sample
  • Complex: Complex of archaeal RNA-polymerase with the ATV inhibitory protein RIP
    • Protein or peptide: x 14 types
  • Ligand: x 3 types
Function / homology
Function and homology information


symbiont-mediated suppression of host transcription / 3 iron, 4 sulfur cluster binding / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / nucleotide binding / DNA-templated transcription ...symbiont-mediated suppression of host transcription / 3 iron, 4 sulfur cluster binding / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / nucleotide binding / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / metal ion binding / cytoplasm
Similarity search - Function
RNA polymerase Rpo13 subunit HTH domain / RNA polymerase Rpo13 subunit HTH domain / DNA-directed RNA polymerase subunit Rpo8 / DNA-directed RNA polymerase, subunit G / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo7 / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo4 ...RNA polymerase Rpo13 subunit HTH domain / RNA polymerase Rpo13 subunit HTH domain / DNA-directed RNA polymerase subunit Rpo8 / DNA-directed RNA polymerase, subunit G / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo7 / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo4 / HRDC domain superfamily / DNA-directed RNA polymerase, subunit E/RPC8 / S1 domain profile. / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / HRDC-like superfamily / RNA polymerase subunit Rpb7-like / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / 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 subunit Rpo11 / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RNA polymerases L / 13 to 16 Kd subunits signature. / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / 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. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / 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 / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit Rpo6 / DNA-directed RNA polymerase subunit Rpo7 / DNA-directed RNA polymerase subunit Rpo3 / DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo11 / RNAP inhibitory protein ...DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit Rpo6 / DNA-directed RNA polymerase subunit Rpo7 / DNA-directed RNA polymerase subunit Rpo3 / DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo11 / RNAP inhibitory protein / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo13 / DNA-directed RNA polymerase subunit Rpo8 / DNA-directed RNA polymerase subunit Rpo4
Similarity search - Component
Biological speciesSulfolobus acidocaldarius DSM 639 (acidophilic) / Acidianus two-tailed virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsPilotto S / Fouqueau T / Lukoyanova N / Sheppard C / Lucas-Staat S / Diaz-Santin LM / Matelska D / Prangishvili D / Cheung ACM / Werner F
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust207446/Z/17/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis of RNA polymerase inhibition by viral and host factors.
Authors: Simona Pilotto / Thomas Fouqueau / Natalya Lukoyanova / Carol Sheppard / Soizick Lucas-Staat / Luis Miguel Díaz-Santín / Dorota Matelska / David Prangishvili / Alan C M Cheung / Finn Werner /
Abstract: RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution ...RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.
History
DepositionJun 2, 2021-
Header (metadata) releaseAug 25, 2021-
Map releaseAug 25, 2021-
UpdateOct 13, 2021-
Current statusOct 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7oq4
  • Surface level: 0.019
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7oq4
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13026.png

Downloads & links

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Map

FileDownload / File: emd_13026.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-process map obtained from Relion 3.0
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 300 pix.
= 314.1 Å		1.05 Å/pix.
x 300 pix.
= 314.1 Å		1.05 Å/pix.
x 300 pix.
= 314.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.047 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.10906489 - 0.19450761
Average (Standard dev.)0.00018650922 (±0.0037748057)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 314.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z314.100314.100314.100
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ332332332
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1090.1950.000

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Supplemental data

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Half map: Half1 map obtained from 3D refinement in Relion...

Fileemd_13026_half_map_1.map
AnnotationHalf1 map obtained from 3D refinement in Relion 3.0. The A/pix has been corrected later during post-processing.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half2 map obtained from 3D refinement in Relion...

Fileemd_13026_half_map_2.map
AnnotationHalf2 map obtained from 3D refinement in Relion 3.0. The A/pix has been corrected later during post-processing.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of archaeal RNA-polymerase with the ATV inhibitory protein RIP

EntireName: Complex of archaeal RNA-polymerase with the ATV inhibitory protein RIP
Components
  • Complex: Complex of archaeal RNA-polymerase with the ATV inhibitory protein RIP
    • Protein or peptide: DNA-directed RNA polymerase subunit A'Polymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit BPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit A''Polymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit DPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit EPolymerase
    • Protein or peptide: DNA-directed RNA polymerase, subunit FPolymerase
    • Protein or peptide: DNA-directed RNA polymerase, subunit GPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit HPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit KPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit LPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit NPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit PPolymerase
    • Protein or peptide: Conserved proteinConservation
    • Protein or peptide: RNAP inhibitory protein
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: FE3-S4 CLUSTER

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Supramolecule #1: Complex of archaeal RNA-polymerase with the ATV inhibitory protein RIP

SupramoleculeName: Complex of archaeal RNA-polymerase with the ATV inhibitory protein RIP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#14
Details: The RNA polymerase was incubated with a 10-fold excess of RIP for 5 min at 338 K followed by cross-linking with BS3
Molecular weightExperimental: 421 KDa

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Macromolecule #1: DNA-directed RNA polymerase subunit A'

MacromoleculeName: DNA-directed RNA polymerase subunit A' / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 99.929055 KDa
SequenceString: MSEKIIRGVK FGVLSPNEIR QMSVTAIITS EVYDEDGTPI EGGVMDPKLG VIEPGQKCPV CGNTLAGCPG HFGHIELIKP VIHIGYVKH IYDFLRSTCW RCGRIKIKEQ DLERYKRIYN AIKLRWPSAA RRLVEYIKKI SIKNLECPHC GEKQFKIKLE K PYNFNEER ...String:
MSEKIIRGVK FGVLSPNEIR QMSVTAIITS EVYDEDGTPI EGGVMDPKLG VIEPGQKCPV CGNTLAGCPG HFGHIELIKP VIHIGYVKH IYDFLRSTCW RCGRIKIKEQ DLERYKRIYN AIKLRWPSAA RRLVEYIKKI SIKNLECPHC GEKQFKIKLE K PYNFNEER NGSIVKLSPS EIRDRLERIP DSDVELLGYD PKSSRPEWMI LTVLPVPPIT IRPSITIESG IRAEDDLTHK LV DIIRLNE RLKESIEAGA PQLIIEDLWD LLQYHVATYF DNEIPGLPPA KHRSGRPLRT LAQRLKGKEG RFRGNLSGKR VDF SARTVI SPDPNLSIDE VGIPYTIARM LTVPERVTNI NIERIRQYII NGPDKWPGAN YVIKPDGRRI DLRYVKDRKE LASS ITAGY VVERHLVDGD VVLFNRQPSL HRISMMAHKV RVLPGRTFRL NLLDCPPYNA DFDGDEMNLH VPQSEEAIAE ARELM LVHK NIITPRYGGP IIGGGQDYIS GAYLLSVKTT LLTVEEVATI LGVTDFVGEL GEPAILAPKP YYTGKQVISL FLPKDF NFH GPANISKGPR ACKDEICPHD SFIVIKNGLL LEGVFDKKAI GNQQPESMLH WSIREYGTEY GKWLMDNVFK MFIRFLE MR GFTMTLEDIT IPDEAQNEIT TKIKEGYSQV DEYIRKFNEG QLEPIPGRTI EESLESYILD TLDKLRKVAG EIATKYLD P FNNVYIMAIT GARGSELNIT QMTALLGQQS VRGERIRRGY RERTLSLFKY GDIAPEARGF VKNSFMRGLS PYEMFFHAA GGREGLVDTA VKTSQSGYMQ RRLINALSDL RIEYDGTVRS LYGDIVQVVY GDDAVHPMYS AHSKSVNVNR VIERVIGWKR

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Macromolecule #2: DNA-directed RNA polymerase subunit B

MacromoleculeName: DNA-directed RNA polymerase subunit B / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 126.647102 KDa
SequenceString: MLDTESRWAI AESFFKTRGL VRQHLDSFND FLRNKLQQVI YEQGEIVTEV PGLKIKLGKI RYEKPSIRET DKGPMREITP MEARLRNLT YSSPIFLSMI PVENNIEGEP IEIYIGDLPI MLKSVADPTS NLPIDKLIEI GEDPKDPGGY FIVNGSEKVI I AQEDLATN ...String:
MLDTESRWAI AESFFKTRGL VRQHLDSFND FLRNKLQQVI YEQGEIVTEV PGLKIKLGKI RYEKPSIRET DKGPMREITP MEARLRNLT YSSPIFLSMI PVENNIEGEP IEIYIGDLPI MLKSVADPTS NLPIDKLIEI GEDPKDPGGY FIVNGSEKVI I AQEDLATN RVLVDYGKSG SNITHVAKVT SSAAGYRVQV MIERLKDSTI QISFATVPGR IPFAIIMRAL GFVTDRDIVY AV SLDPQIQ NELLPSLEQA SSITSAEEAL DFIGNRVAIG QKRENRIQKA EQVIDKYFLP HLGTSPEDRK KKGYYLASAV NKI LELYLG RREPDDKDHY ANKRVRLAGD LFTSLFRVAF KAFVKDLVYQ LEKSKVRGRR LSLTALVRAD IITERIRHAL ATGN WVGGR TGVSQLLDRT NWLSMLSHLR RVVSSLARGQ PNFEARDLHG TQWGRMCPFE TPEGPNSGLV KNLALLAQVS VGINE SVVE RVAYELGVVS VEDVIRRISE QNEDVEKYMS WSKVYLNGRL LGYYEDGKEL AKKIRESRRQ GKLSDEVNVA YIATDY LNE VHINCDAGRV RRPLIIVNNG TPLVDTEDIK KLKNGEITFD DLVKQGKIEF IDAEEEENAY VALNPQDLTP DHTHLEI WP SAILGIIASI IPYPEHNQSP RNTYQSAMAK QSLGLYASNY QIRTDTRAHL LHYPQMPLVQ TRMLGVIGYN DRPAGANA I LAIMSYTGYN MEDSIIMNKS SIERGMYRST FFRLYSTEEV KYPGGQEDKI ITPEAGVKGY KGKDYYRLLE DNGVVSPEV EVKGGDVLIG KVSPPRFLQE FKELSPEQAK RDTSIVTRHG ENGIVDLVLI TETLEGNKLV KVRVRDLRIP EIGDKFATRH GQKGVVGIL IDQVDMPYTA KGIVPDIILN PHALPSRMTI GQIMEAIGGK YAALSGKPVD ATPFLETPKL QEMQKEILKL G HLPDSTEV VYDGRTGQKL KSRILFGIVY YQKLHHMVAD KMHARARGPV QILTRQPTEG RAREGGLRFG EMERDCLIGF GT AMLIKDR LLDNSDKAVV YICDQCGYVG WYDRSKNRYV CPVHGDKSVL HPVTVSYAFK LLIQELMSMV ISPRLILGEK VNL GGASNE

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Macromolecule #3: DNA-directed RNA polymerase subunit A''

MacromoleculeName: DNA-directed RNA polymerase subunit A'' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 44.50743 KDa
SequenceString: MIDEKLKGYI DKRLNEIKDK IPDKLHEDLR AAIMDINGVE LTEEDIDRII DLTIREYQQS LIEPGEAIGV VTAQSVGEPG TQMTLRTFH FAGIRELNVT LGLPRLIEIV DARKVPSTPM MTIYLTDEYK TDKDKALDIA RRIEYTRVEN VVSSVSVDIS N MSITLQFD ...String:
MIDEKLKGYI DKRLNEIKDK IPDKLHEDLR AAIMDINGVE LTEEDIDRII DLTIREYQQS LIEPGEAIGV VTAQSVGEPG TQMTLRTFH FAGIRELNVT LGLPRLIEIV DARKVPSTPM MTIYLTDEYK TDKDKALDIA RRIEYTRVEN VVSSVSVDIS N MSITLQFD QEMLKDKGVS IEEIKKIITK LKLGEIRIED NDEYSFTIYF EKIDSIMALF KMREKILNTK IKGVKGIKRA IV QKKGDEY VIITDGSNLE GIMNVTGVDI NKIQTNNIHE VEEVLGIEAA RELISREIKK VLEEQGLDVD MRHIVLVSDI MTR TGDIRQ IGRHGVTGEK SSVLARAAFE VTVKHLLDAA ARGEREEFKG VIENIIIGQP IRLGTGIVEL TMKPNMR

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Macromolecule #4: DNA-directed RNA polymerase subunit D

MacromoleculeName: DNA-directed RNA polymerase subunit D / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 29.858752 KDa
SequenceString: MPISLIERNG LRLRLVLENY PLEFVNSIRR ASILYVPVMA VDEVYFIENN SPLYDEILAH RLALVPFVSD EALEHYRPPE ECAECKENC DGCYNRVYLD VEAKDQPLMI YSRDLKSEDQ MITPVSGAIP IVLLGSKQKI SLEARLRLGY GKEHIKYSPV S VSIVRYYP ...String:
MPISLIERNG LRLRLVLENY PLEFVNSIRR ASILYVPVMA VDEVYFIENN SPLYDEILAH RLALVPFVSD EALEHYRPPE ECAECKENC DGCYNRVYLD VEAKDQPLMI YSRDLKSEDQ MITPVSGAIP IVLLGSKQKI SLEARLRLGY GKEHIKYSPV S VSIVRYYP KVTVLGNCEK AVEVCPEGVF AMENNKLVVK NELSCILCEE CLKYCAGSVS IESVENKFIL EIESVGSLKP ER ILIEASK SLLRKLSELK SKLEAGK

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Macromolecule #5: DNA-directed RNA polymerase subunit E

MacromoleculeName: DNA-directed RNA polymerase subunit E / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 20.49675 KDa
SequenceString:
MFKLVRAKGI VRIPPEYFGQ SVDEIAIKIL RQEYQEKLIK DIGVVLGIVN AKASEEGFII FGDGATYHEV EFDMLVYTPI IHEVIEGEV SQVDNYGVYV NMGPVDGLVH ISQITDDNLK FDSNRGILIG EKSKKSIQKG DRVRAMIISA SMSSGRLPRI A LTMKQPYL GKIEWINQEI AKASK

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Macromolecule #6: DNA-directed RNA polymerase, subunit F

MacromoleculeName: DNA-directed RNA polymerase, subunit F / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 13.070987 KDa
SequenceString:
MSYSLKTIEE HFVPYSIAKK YIKELIDTGS SSNLIQKTFD YLNSISRCDE DSASKIMKEL EEIVKREDVR AVLASICPTT VEEVRSVLV IDPSTIYSTE QIQKIIEIIK KYVES

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Macromolecule #7: DNA-directed RNA polymerase, subunit G

MacromoleculeName: DNA-directed RNA polymerase, subunit G / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 15.292758 KDa
Recombinant expressionOrganism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
SequenceString:
MKEMMQGTCK ISSIEKGALK NLYVVKMDCD NDLKIEFDIT KELSIFSKDE EVTFIISREK PEYSEKDFCA HGYLFLERQQ EDGSFIDEI SLYGLIVKIL SKNGLINSKL FKMMDHVYYC VKKKAHHHHH H

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Macromolecule #8: DNA-directed RNA polymerase subunit H

MacromoleculeName: DNA-directed RNA polymerase subunit H / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 9.477059 KDa
SequenceString:
MRSSSKKKID ISNHELVPKH EILQLEEAYK LVKELGIKPE QLPWIRASDP VAKSIGAKPG DIIKITRKSP FTGESVTYRY VITG

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Macromolecule #9: DNA-directed RNA polymerase subunit K

MacromoleculeName: DNA-directed RNA polymerase subunit K / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 10.25199 KDa
SequenceString:
MTIDKINEIF KENWKNKLTK YEIARIISAR ALQLSMGALP LIDTSNLKSD DVISIAEEEL KRGVLPITIR RIYPNGQVEL ISVRKIENR

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Macromolecule #10: DNA-directed RNA polymerase subunit L

MacromoleculeName: DNA-directed RNA polymerase subunit L / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 10.061791 KDa
SequenceString:
MEIKVIKEEQ NYLELQIDGE EHTIGNLLKG MLLKVPGVKF AAYSLPHPLI TSITIKILTD GSISAREALI KAIELAENYA NLFIDEVKK I

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Macromolecule #11: DNA-directed RNA polymerase subunit N

MacromoleculeName: DNA-directed RNA polymerase subunit N / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 7.673108 KDa
SequenceString:
MIIPIRCFTC GAVVADKWEP FSNRVMGGED PEKVLTELGV NRYCCRRMLL SHVNIIREII HYTRPI

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Macromolecule #12: DNA-directed RNA polymerase subunit P

MacromoleculeName: DNA-directed RNA polymerase subunit P / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 5.662912 KDa
SequenceString:
MAKYRCGKCW KELDDDQLKT LPGVRCPYCG YRIIYMVRKP TVKIVKAI

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Macromolecule #13: Conserved protein

MacromoleculeName: Conserved protein / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Molecular weightTheoretical: 12.331662 KDa
SequenceString:
MSEDDSKKEP EPEETEAEIK HEEISREEDD EGGEFSTVTI SDIEMLLKDT EIWDKLLRNE LSIEEAKKMF DDVARSYSKA DKKKRRVEK KPKKGKVTKK SDEEEE

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Macromolecule #14: RNAP inhibitory protein

MacromoleculeName: RNAP inhibitory protein / type: protein_or_peptide / ID: 14
Details: Residual sequence from the thrombin cleavage site at the C-terminus of RIP (LVPR)
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Acidianus two-tailed virus
Molecular weightTheoretical: 17.321936 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MKNMLHPQKY ETHVLDDLME FYEGVIGYPE IDLRLAGEEA WLKGVNPELA EAVKKIIKTI RRYLEGSPYD GSEKPIPRYI IAEIFSQIA PEVQLLVNAL DTEGKYGFLK HIKKLNLNSL AMLSKNYNEN DKLWKELENE GYVYLELVPR

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Macromolecule #15: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 15 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #16: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 16 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #17: FE3-S4 CLUSTER

MacromoleculeName: FE3-S4 CLUSTER / type: ligand / ID: 17 / Number of copies: 1 / Formula: F3S
Molecular weightTheoretical: 295.795 Da
Chemical component information

ChemComp-F3S:
FE3-S4 CLUSTER / Iron–sulfur cluster

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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

Concentration0.06 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4S4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
200.0 mMNaClSodium chloridesodium chloride
5.0 mMMgCl2magnesium chloride
100.0 microMZnSO4zinc sulphate
10.0 % w/vC3H8O3glycerol
5.0 mMC4H10O2S2dithiothreitol

Details: DTT was added fresh before the incubation of the two species
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: after glow discharged, the grid was coated with graphene oxide
VitrificationCryogen name: ETHANE / Chamber humidity: 94 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: blotting force: -10 blotting time: 2 sec wait time: 0 sec.
DetailsThe sample was monodisperse

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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 / Cs: 2.7 mm / Nominal defocus max: -2.5 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Detailssample holder was tilted of -30 degrees for the entire data collection
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-45 / Number grids imaged: 1 / Number real images: 2130 / Average electron dose: 45.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 600640
CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 2.0) / Software - details: 2D classification
Final 3D classificationNumber classes: 2 / Avg.num./class: 75000 / Software - Name: cryoSPARC (ver. 2.0) / Software - details: 3D reconstruction
Details: The classification provided two classes of identical maps, splitting the particles equally in the two classes. For that, the particles of both classes have been pooled together for the final reconstruction
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 2.0) / Software - details: 3D reconstruction
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Software - details: 3D refinement, followed by three cycles of CTF refinement and post processing
Number images used: 151237
DetailsThe movie-stacks were aligned and summed using MotionCor2 within Relion 3.0
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7oh0


3faj

chain_id: A
DetailsThe C-terminal tail of RIP was manually built in Coot after the first refinement run.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 81.4405 / Target criteria: correlation coefficient
Output model

PDB-7oq4:
Cryo-EM structure of the ATV RNAP Inhibitory Protein (RIP) bound to the DNA-binding channel of the host's RNA polymerase

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