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- EMDB-15357: RNA polymerase at U-rich pause bound to RNA putL triple mutant - ... -

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Basic information

Entry
Database: EMDB / ID: EMD-15357
TitleRNA polymerase at U-rich pause bound to RNA putL triple mutant - pause prone, closed clamp state
Map data
Sample
  • Complex: Escherichia coli RNA polymerase putL triple mutant complex - pause prone, closed clamp state
    • DNA: Non-template DNA
    • RNA: RNA putL triple mutant
    • DNA: Template DNA
    • Protein or peptide: DNA-directed RNA polymerase subunit omega
    • Protein or peptide: RNA polymerase sigma factor RpoD
    • Protein or peptide: DNA-directed RNA polymerase subunit alpha
    • Protein or peptide: DNA-directed RNA polymerase subunit beta
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
KeywordsRNA polymerase / transcriptional pausing / transcription termination / regulatory RNA / TRANSCRIPTION
Function / homology
Function and homology information


sigma factor antagonist complex / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / bacterial-type RNA polymerase core enzyme binding / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility ...sigma factor antagonist complex / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / bacterial-type RNA polymerase core enzyme binding / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / DNA-directed RNA polymerase complex / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / negative regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 ...RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / 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 / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsDey S / Weixlbaumer A
Funding supportEuropean Union, France, 4 items
OrganizationGrant numberCountry
European Research Council (ERC)679734European Union
French Infrastructure for Integrated Structural Biology (FRISBI)ANR-10-INBS-05 France
Agence Nationale de la Recherche (ANR)ANR-10-LABX-0030-INRT France
Agence Nationale de la Recherche (ANR)ANR-10-IDEX-0002-02 France
CitationJournal: Mol Cell / Year: 2022
Title: Structural insights into RNA-mediated transcription regulation in bacteria.
Authors: Sanjay Dey / Claire Batisse / Jinal Shukla / Michael W Webster / Maria Takacs / Charlotte Saint-André / Albert Weixlbaumer /
Abstract: RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at ...RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.
History
DepositionJul 7, 2022-
Header (metadata) releaseOct 19, 2022-
Map releaseOct 19, 2022-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15357.png

Downloads & links

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Map

FileDownload / File: emd_15357.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 360 pix.
= 310.32 Å		0.86 Å/pix.
x 360 pix.
= 310.32 Å		0.86 Å/pix.
x 360 pix.
= 310.32 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.862 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.118
Minimum - Maximum-0.16610187 - 0.43561068
Average (Standard dev.)0.0021547626 (±0.018731583)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 310.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_15357_additional_1.map
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Half map: #2

Fileemd_15357_half_map_1.map
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Half map: #1

Fileemd_15357_half_map_2.map
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Sample components

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Entire : Escherichia coli RNA polymerase putL triple mutant complex - paus...

EntireName: Escherichia coli RNA polymerase putL triple mutant complex - pause prone, closed clamp state
Components
  • Complex: Escherichia coli RNA polymerase putL triple mutant complex - pause prone, closed clamp state
    • DNA: Non-template DNA
    • RNA: RNA putL triple mutant
    • DNA: Template DNA
    • Protein or peptide: DNA-directed RNA polymerase subunit omega
    • Protein or peptide: RNA polymerase sigma factor RpoD
    • Protein or peptide: DNA-directed RNA polymerase subunit alpha
    • Protein or peptide: DNA-directed RNA polymerase subunit beta
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Escherichia coli RNA polymerase putL triple mutant complex - paus...

SupramoleculeName: Escherichia coli RNA polymerase putL triple mutant complex - pause prone, closed clamp state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 435 kDa/nm

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Macromolecule #1: Non-template DNA

MacromoleculeName: Non-template DNA / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 82.140438 KDa
SequenceString: (DC)(DA)(DG)(DT)(DC)(DA)(DC)(DG)(DA)(DC) (DG)(DT)(DT)(DG)(DT)(DA)(DA)(DA)(DA)(DC) (DG)(DA)(DC)(DG)(DG)(DC)(DC)(DA)(DG) (DT)(DG)(DA)(DA)(DT)(DT)(DC)(DG)(DA)(DG) (DC) (DT)(DC)(DG)(DG)(DT)(DA) ...String:
(DC)(DA)(DG)(DT)(DC)(DA)(DC)(DG)(DA)(DC) (DG)(DT)(DT)(DG)(DT)(DA)(DA)(DA)(DA)(DC) (DG)(DA)(DC)(DG)(DG)(DC)(DC)(DA)(DG) (DT)(DG)(DA)(DA)(DT)(DT)(DC)(DG)(DA)(DG) (DC) (DT)(DC)(DG)(DG)(DT)(DA)(DC)(DC) (DA)(DA)(DA)(DA)(DA)(DT)(DA)(DA)(DA)(DT) (DT)(DT) (DC)(DC)(DT)(DT)(DA)(DA)(DA) (DG)(DT)(DT)(DC)(DA)(DC)(DT)(DA)(DA)(DC) (DT)(DT)(DA) (DT)(DG)(DA)(DT)(DG)(DT) (DA)(DG)(DT)(DG)(DA)(DG)(DC)(DT)(DT)(DT) (DT)(DT)(DA)(DT) (DA)(DC)(DC)(DC)(DA) (DT)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DA)(DC) (DT)(DA)(DT)(DT)(DG) (DG)(DT)(DA)(DC) (DT)(DT)(DT)(DA)(DC)(DA)(DT)(DT)(DA)(DA) (DT)(DG)(DA)(DA)(DC)(DT) (DT)(DT)(DA) (DA)(DG)(DT)(DA)(DC)(DA)(DT)(DC)(DA)(DT) (DA)(DA)(DG)(DC)(DC)(DC)(DA) (DT)(DA) (DG)(DA)(DC)(DG)(DA)(DA)(DC)(DG)(DG)(DC) (DC)(DC)(DG)(DT)(DC)(DT)(DT)(DT) (DA) (DA)(DA)(DC)(DC)(DA)(DT)(DG)(DC)(DG)(DT) (DC)(DG)(DG)(DG)(DT)(DG)(DC)(DC)(DC) (DG)(DG)(DC)(DG)(DG)(DG)(DT)(DT)(DC)(DA) (DG)(DG)(DA)(DT)(DG)(DA)(DA)(DC)(DG)(DG) (DC)(DA)(DA)(DT)(DG)(DC)(DT)(DG)(DC) (DT)(DC)(DA)(DT)(DT)(DA)(DG)(DC)(DG)(DA) (DG) (DA)(DA)(DG)(DG)(DC)(DT)(DT)(DT) (DT)(DT)(DT)(DG)(DT)(DT)(DT)(DT)(DT)(DA) (DG)(DT) (DC)(DA)(DC)(DG)(DG)(DC)

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Macromolecule #3: Template DNA

MacromoleculeName: Template DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 82.141461 KDa
SequenceString: (DG)(DC)(DC)(DG)(DT)(DG)(DA)(DC)(DT)(DA) (DA)(DA)(IGU)(IGU)(DC)(DA)(DA)(DA)(DA) (DA)(DA)(DG)(DC)(DC)(DT)(DT)(DC)(DT)(DC) (DG)(DC)(DT)(DA)(DA)(DT)(DG)(DA)(DG)(DC) (DA)(DG)(DC)(DA)(DT)(DT)(DG) ...String:
(DG)(DC)(DC)(DG)(DT)(DG)(DA)(DC)(DT)(DA) (DA)(DA)(IGU)(IGU)(DC)(DA)(DA)(DA)(DA) (DA)(DA)(DG)(DC)(DC)(DT)(DT)(DC)(DT)(DC) (DG)(DC)(DT)(DA)(DA)(DT)(DG)(DA)(DG)(DC) (DA)(DG)(DC)(DA)(DT)(DT)(DG)(DC)(DC) (DG)(DT)(DT)(DC)(DA)(DT)(DC)(DC)(DT)(DG) (DA) (DA)(DC)(DC)(DC)(DG)(DC)(DC)(DG) (DG)(DG)(DC)(DA)(DC)(DC)(DC)(DG)(DA)(DC) (DG)(DC) (DA)(DT)(DG)(DG)(DT)(DT)(DT) (DA)(DA)(DA)(DG)(DA)(DC)(DG)(DG)(DG)(DC) (DC)(DG)(DT) (DT)(DC)(DG)(DT)(DC)(DT) (DA)(DT)(DG)(DG)(DG)(DC)(DT)(DT)(DA)(DT) (DG)(DA)(DT)(DG) (DT)(DA)(DC)(DT)(DT) (DA)(DA)(DA)(DG)(DT)(DT)(DC)(DA)(DT)(DT) (DA)(DA)(DT)(DG)(DT) (DA)(DA)(DA)(DG) (DT)(DA)(DC)(DC)(DA)(DA)(DT)(DA)(DG)(DT) (DA)(DC)(DA)(DT)(DT)(DT) (DT)(DA)(DT) (DG)(DG)(DG)(DT)(DA)(DT)(DA)(DA)(DA)(DA) (DA)(DG)(DC)(DT)(DC)(DA)(DC) (DT)(DA) (DC)(DA)(DT)(DC)(DA)(DT)(DA)(DA)(DG)(DT) (DT)(DA)(DG)(DT)(DG)(DA)(DA)(DC) (DT) (DT)(DT)(DA)(DA)(DG)(DG)(DA)(DA)(DA)(DT) (DT)(DT)(DA)(DT)(DT)(DT)(DT)(DT)(DG) (DG)(DT)(DA)(DC)(DC)(DG)(DA)(DG)(DC)(DT) (DC)(DG)(DA)(DA)(DT)(DT)(DC)(DA)(DC)(DT) (DG)(DG)(DC)(DC)(DG)(DT)(DC)(DG)(DT) (DT)(DT)(DT)(DA)(DC)(DA)(DA)(DC)(DG)(DT) (DC) (DG)(DT)(DG)(DA)(DC)(DT)(DG)

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Macromolecule #2: RNA putL triple mutant

MacromoleculeName: RNA putL triple mutant / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 29.999773 KDa
SequenceString:
AUAGACGAAC GGCCCGUCUU UAAACCAUGC GUCGGGUGCC CGGCGGGUUC AGGAUGAACG GCAAUGCUGC UCAUUAGCGA GAAGGCUUU UUUG

GENBANK: GENBANK: CP053852.1

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Macromolecule #4: DNA-directed RNA polymerase subunit omega

MacromoleculeName: DNA-directed RNA polymerase subunit omega / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 10.249547 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MARVTVQDAV EKIGNRFDLV LVAARRARQM QVGGKDPLVP EENDKTTVIA LREIEEGLIN NQILDVRERQ EQQEQEAAEL QAVTAIAEG RR

UniProtKB: DNA-directed RNA polymerase subunit omega

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Macromolecule #5: RNA polymerase sigma factor RpoD

MacromoleculeName: RNA polymerase sigma factor RpoD / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
Molecular weightTheoretical: 70.327219 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MEQNPQSQLK LLVTRGKEQG YLTYAEVNDH LPEDIVDSDQ IEDIIQMIND MGIQVMEEAP DADDLMLAEN TADEDAAEAA AQVLSSVES EIGRTTDPVR MYMREMGTVE LLTREGEIDI AKRIEDGINQ VQCSVAEYPE AITYLLEQYD RVEAEEARLS D LITGFVDP ...String:
MEQNPQSQLK LLVTRGKEQG YLTYAEVNDH LPEDIVDSDQ IEDIIQMIND MGIQVMEEAP DADDLMLAEN TADEDAAEAA AQVLSSVES EIGRTTDPVR MYMREMGTVE LLTREGEIDI AKRIEDGINQ VQCSVAEYPE AITYLLEQYD RVEAEEARLS D LITGFVDP NAEEDLAPTA THVGSELSQE DLDDDEDEDE EDGDDDSADD DNSIDPELAR EKFAELRAQY VVTRDTIKAK GR SHATAQE EILKLSEVFK QFRLVPKQFD YLVNSMRVMM DRVRTQERLI MKLCVEQCKM PKKNFITLFT GNETSDTWFN AAI AMNKPW SEKLHDVSEE VHRALQKLQQ IEEETGLTIE QVKDINRRMS IGEAKARRAK KEMVEANLRL VISIAKKYTN RGLQ FLDLI QEGNIGLMKA VDKFEYRRGY KFSTYATWWI RQAITRSIAD QARTIRIPVH MIETINKLNR ISRQMLQEMG REPTP EELA ERMLMPEDKI RKVLKIAKEP ISMETPIGDD EDSHLGDFIE DTTLELPLDS ATTESLRAAT HDVLAGLTAR EAKVLR MRF GIDMNTDHTL EEVGKQFDVT RERIRQIEAK ALRKLRHPSR SEVLRSFLDD

UniProtKB: RNA polymerase sigma factor RpoD

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Macromolecule #6: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 36.55868 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MQGSVTEFLK PRLVDIEQVS STHAKVTLEP LERGFGHTLG NALRRILLSS MPGCAVTEVE IDGVLHEYST KEGVQEDILE ILLNLKGLA VRVQGKDEVI LTLNKSGIGP VTAADITHDG DVEIVKPQHV ICHLTDENAS ISMRIKVQRG RGYVPASTRI H SEEDERPI ...String:
MQGSVTEFLK PRLVDIEQVS STHAKVTLEP LERGFGHTLG NALRRILLSS MPGCAVTEVE IDGVLHEYST KEGVQEDILE ILLNLKGLA VRVQGKDEVI LTLNKSGIGP VTAADITHDG DVEIVKPQHV ICHLTDENAS ISMRIKVQRG RGYVPASTRI H SEEDERPI GRLLVDACYS PVERIAYNVE AARVEQRTDL DKLVIEMETN GTIDPEEAIR RAATILAEQL EAFVDLRDVR QP EVKEEKP EFDPILLRPV DDLELTVRSA NCLKAEAIHY IGDLVQRTEV ELLKTPNLGK KSLTEIKDVL ASRGLSLGMR LEN WPPASI ADE

UniProtKB: DNA-directed RNA polymerase subunit alpha

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Macromolecule #7: DNA-directed RNA polymerase subunit beta

MacromoleculeName: DNA-directed RNA polymerase subunit beta / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 150.820875 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MVYSYTEKKR IRKDFGKRPQ VLDVPYLLSI QLDSFQKFIE QDPEGQYGLE AAFRSVFPIQ SYSGNSELQY VSYRLGEPVF DVQECQIRG VTYSAPLRVK LRLVIYEREA PEGTVKDIKE QEVYMGEIPL MTDNGTFVIN GTERVIVSQL HRSPGVFFDS D KGKTHSSG ...String:
MVYSYTEKKR IRKDFGKRPQ VLDVPYLLSI QLDSFQKFIE QDPEGQYGLE AAFRSVFPIQ SYSGNSELQY VSYRLGEPVF DVQECQIRG VTYSAPLRVK LRLVIYEREA PEGTVKDIKE QEVYMGEIPL MTDNGTFVIN GTERVIVSQL HRSPGVFFDS D KGKTHSSG KVLYNARIIP YRGSWLDFEF DPKDNLFVRI DRRRKLPATI ILRALNYTTE QILDLFFEKV IFEIRDNKLQ ME LVPERLR GETASFDIEA NGKVYVEKGR RITARHIRQL EKDDVKLIEV PVEYIAGKVV AKDYIDESTG ELICAANMEL SLD LLAKLS QSGHKRIETL FTNDLDHGPY ISETLRVDPT NDRLSALVEI YRMMRPGEPP TREAAESLFE NLFFSEDRYD LSAV GRMKF NRSLLREEIE GSGILSKDDI IDVMKKLIDI RNGKGEVDDI DHLGNRRIRS VGEMAENQFR VGLVRVERAV KERLS LGDL DTLMPQDMIN AKPISAAVKE FFGSSQLSQF MDQNNPLSEI THKRRISALG PGGLTRERAG FEVRDVHPTH YGRVCP IET PEGPNIGLIN SLSVYAQTNE YGFLETPYRK VTDGVVTDEI HYLSAIEEGN YVIAQANSNL DEEGHFVEDL VTCRSKG ES SLFSRDQVDY MDVSTQQVVS VGASLIPFLE HDDANRALMG ANMQRQAVPT LRADKPLVGT GMERAVAVDS GVTAVAKR G GVVQYVDASR IVIKVNEDEM YPGEAGIDIY NLTKYTRSNQ NTCINQMPCV SLGEPVERGD VLADGPSTDL GELALGQNM RVAFMPWNGY NFEDSILVSE RVVQEDRFTT IHIQELACVS RDTKLGPEEI TADIPNVGEA ALSKLDESGI VYIGAEVTGG DILVGKVTP KGETQLTPEE KLLRAIFGEK ASDVKDSSLR VPNGVSGTVI DVQVFTRDGV EKDKRALEIE EMQLKQAKKD L SEELQILE AGLFSRIRAV LVAGGVEAEK LDKLPRDRWL ELGLTDEEKQ NQLEQLAEQY DELKHEFEKK LEAKRRKITQ GD DLAPGVL KIVKVYLAVK RRIQPGDKMA GRHGNKGVIS KINPIEDMPY DENGTPVDIV LNPLGVPSRM NIGQILETHL GMA AKGIGD KINAMLKQQQ EVAKLREFIQ RAYDLGADVR QKVDLSTFSD EEVMRLAENL RKGMPIATPV FDGAKEAEIK ELLK LGDLP TSGQIRLYDG RTGEQFERPV TVGYMYMLKL NHLVDDKMHA RSTGSYSLVT QQPLGGKAQF GGQRFGEMEV WALEA YGAA YTLQEMLTVK SDDVNGRTKM YKNIVDGNHQ MEPGMPESFN VLLKEIRSLG INIELEDE

UniProtKB: DNA-directed RNA polymerase subunit beta

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Macromolecule #8: DNA-directed RNA polymerase subunit beta'

MacromoleculeName: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 155.237672 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKDLLKFLKA QTKTEEFDAI KIALASPDMI RSWSFGEVKK PETINYRTFK PERDGLFCAR IFGPVKDYEC LCGKYKRLKH RGVICEKCG VEVTQTKVRR ERMGHIELAS PTAHIWFLKS LPSRIGLLLD MPLRDIERVL YFESYVVIEG GMTNLERQQI L TEEQYLDA ...String:
MKDLLKFLKA QTKTEEFDAI KIALASPDMI RSWSFGEVKK PETINYRTFK PERDGLFCAR IFGPVKDYEC LCGKYKRLKH RGVICEKCG VEVTQTKVRR ERMGHIELAS PTAHIWFLKS LPSRIGLLLD MPLRDIERVL YFESYVVIEG GMTNLERQQI L TEEQYLDA LEEFGDEFDA KMGAEAIQAL LKSMDLEQEC EQLREELNET NSETKRKKLT KRIKLLEAFV QSGNKPEWMI LT VLPVLPP DLRPLVPLDG GRFATSDLND LYRRVINRNN RLKRLLDLAA PDIIVRNEKR MLQEAVDALL DNGRRGRAIT GSN KRPLKS LADMIKGKQG RFRQNLLGKR VDYSGRSVIT VGPYLRLHQC GLPKKMALEL FKPFIYGKLE LRGLATTIKA AKKM VEREE AVVWDILDEV IREHPVLLNR APTLHRLGIQ AFEPVLIEGK AIQLHPLVCA AYNADFDGDQ MAVHVPLTLE AQLEA RALM MSTNNILSPA NGEPIIVPSQ DVVLGLYYMT RDCVNAKGEG MVLTGPKEAE RLYRSGLASL HARVKVRITE YEKDAN GEL VAKTSLKDTT VGRAILWMIV PKGLPYSIVN QALGKKAISK MLNTCYRILG LKPTVIFADQ IMYTGFAYAA RSGASVG ID DMVIPEKKHE IISEAEAEVA EIQEQFQSGL VTAGERYNKV IDIWAAANDR VSKAMMDNLQ TETVINRDGQ EEKQVSFN S IYMMADSGAR GSAAQIRQLA GMRGLMAKPD GSIIETPITA NFREGLNVLQ YFISTHGARK GLADTALKTA NSGYLTRRL VDVAQDLVVT EDDCGTHEGI MMTPVIEGGD VKEPLRDRVL GRVTAEDVLK PGTADILVPR NTLLHEQWCD LLEENSVDAV KVRSVVSCD TDFGVCAHCY GRDLARGHII NKGEAIGVIA AQSIGEPGTQ LTMRTFHIGG AASRAAAESS IQVKNKGSIK L SNVKSVVN SSGKLVITSR NTELKLIDEF GRTKESYKVP YGAVLAKGDG EQVAGGETVA NWDPHTMPVI TEVSGFVRFT DM IDGQTIT RQTDELTGLS SLVVLDSAER TAGGKDLRPA LKIVDAQGND VLIPGTDMPA QYFLPGKAIV QLEDGVQISS GDT LARIPQ ESGGTKDITG GLPRVADLFE ARRPKEPAIL AEISGIVSFG KETKGKRRLV ITPVDGSDPY EEMIPKWRQL NVFE GERVE RGDVISDGPE APHDILRLRG VHAVTRYIVN EVQDVYRLQG VKINDKHIEV IVRQMLRKAT IVNAGSSDFL EGEQV EYSR VKIANRELEA NGKVGATYSR DLLGITKASL ATESFISAAS FQETTRVLTE AAVAGKRDEL RGLKENVIVG RLIPAG TGY AYHQDRMRRR AAGEAPAAPQ VTAEDASASL AELLNAGLGG SDN

UniProtKB: DNA-directed RNA polymerase subunit beta'

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 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

Concentration12 mg/mL
BufferpH: 8
Details: 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, 2mM DTT
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Details: Quantifoil UltrAuFoil R1.2/1.3 300 mesh holey gold grids were plasma cleaned on a Model 1070 (Fischione Instruments) for 30 sec at 70% power and with an 80% Argon and 20% Oxygen mixture ...Details: Quantifoil UltrAuFoil R1.2/1.3 300 mesh holey gold grids were plasma cleaned on a Model 1070 (Fischione Instruments) for 30 sec at 70% power and with an 80% Argon and 20% Oxygen mixture prior to the application of 0.004 ml of sample. Grids were plunge frozen into liquid ethane using a Vitrobot mark IV (FEI) with 95% chamber humidity at 283K..
DetailsThe co-transcriptionally halted complexes for cryo-EM analysis were prepared in vitro by mixing E. coli RNAP holoenzyme with templates containing the DNA base analogue isoG. The RNAP ECs halted at the U-rich pause (G93). The complexes were prepared in 0.03 ml reactions and contained: 0.02 mM of E. coli RNAP, 0.080 mM of E. coli Sigma70, 0.02 mM of template DNA, 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, 2mM DTT and 6 mM of each ATP, GTP, CTP and UTP. Initially, all the components except NTPs were added and incubated for 10 min at 310K. To allow transcription elongation, NTPs were added and the sample was incubated for 20 to 40 min at 310K. The final products were purified on a Superose 6 Increase 3.2/300 gel filtration column equilibrated in Glutamate buffer. Samples were concentrated to 10-12 mg/mL using an Amicon Ultra 0.5mL centrifugal filter unit (30 KDa MWCO). Before grid freezing, 8 mM of CHAPSO was added to freshly prepared sample to overcome preferred particle orientation.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average exposure time: 2.2 sec. / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 93293
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3)

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Atomic model buiding 1

Initial modelPDB ID:

6alh


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: map cross correlation
Output model

PDB-8ad1:
RNA polymerase at U-rich pause bound to RNA putL triple mutant - pause prone, closed clamp state

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