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- EMDB-17685: E. coli transcription complex paused at ops site and bound to Rfa... -

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Basic information

Entry
Database: EMDB / ID: EMD-17685
TitleE. coli transcription complex paused at ops site and bound to RfaH and NusA
Map data
Sample
  • Complex: transcription complex paused at ops site, assembled on a fully complementary nucleic acid scaffold, bound to RfaH and NusA
    • Complex: Transcription complex
      • Protein or peptide: x 6 types
    • Complex: DNA and RNA
      • DNA: x 2 types
      • RNA: x 1 types
  • Ligand: x 2 types
Keywordstranscription complex / ops / RfaH / NusA / TRANSCRIPTION
Function / homology
Function and homology information


regulatory RNA binding / transcription antitermination factor activity, DNA binding / translation activator activity / DNA-templated transcription elongation / bacterial-type RNA polymerase core enzyme binding / transcription elongation-coupled chromatin remodeling / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex ...regulatory RNA binding / transcription antitermination factor activity, DNA binding / translation activator activity / DNA-templated transcription elongation / bacterial-type RNA polymerase core enzyme binding / transcription elongation-coupled chromatin remodeling / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / positive regulation of translation / regulation of DNA-templated transcription elongation / transcription antitermination / cell motility / DNA-templated transcription termination / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / ribosome biogenesis / protein complex oligomerization / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / DNA-binding transcription factor activity / protein domain specific binding / response to antibiotic / nucleotide binding / magnesium ion binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Transcription antitermination protein RfaH / Transcription termination factor NusA, C-terminal duplication / Transcription termination factor NusA / Transcription factor NusA, N-terminal / KH domain, NusA-like / NusA, N-terminal domain superfamily / NusA N-terminal domain / NusA-like KH domain / Transcription termination/antitermination protein NusA, bacterial / NusG-like ...Transcription antitermination protein RfaH / Transcription termination factor NusA, C-terminal duplication / Transcription termination factor NusA / Transcription factor NusA, N-terminal / KH domain, NusA-like / NusA, N-terminal domain superfamily / NusA N-terminal domain / NusA-like KH domain / Transcription termination/antitermination protein NusA, bacterial / NusG-like / Transcription termination factor nusG / NusG, N-terminal / In Spt5p, this domain may confer affinity for Spt4p. It possesses a RNP-like fold. / NusG, N-terminal domain superfamily / Type-1 KH domain profile. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain / S1 domain profile. / 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 / 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, domain 3 superfamily / 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 / K homology domain superfamily, prokaryotic type / K homology domain-like, alpha/beta / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Transcription termination/antitermination protein NusA / Transcription antitermination protein RfaH
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZuber PK / Said N / Hilal T / Loll B / Wahl MC / Knauer SH
Funding support Germany, 3 items
OrganizationGrant numberCountry
German Research Foundation (DFG)WA 1126/11-1 Germany
German Research Foundation (DFG)INST 130/1064-1 Germany
German Research Foundation (DFG)Ro617/21-1 Germany
CitationJournal: Nat Commun / Year: 2024
Title: Concerted transformation of a hyper-paused transcription complex and its reinforcing protein.
Authors: Philipp K Zuber / Nelly Said / Tarek Hilal / Bing Wang / Bernhard Loll / Jorge González-Higueras / César A Ramírez-Sarmiento / Georgiy A Belogurov / Irina Artsimovitch / Markus C Wahl / Stefan H Knauer /
Abstract: RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an α-helical KOW domain ...RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an α-helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a β-barrel, which binds the ribosome. Here, we report structures of ops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH, which reveal swiveled, pre-translocated pause states stabilized by an ops hairpin in the non-template DNA. Autoinhibited RfaH binds and twists the ops hairpin, expanding the RNA:DNA hybrid to 11 base pairs and triggering the KOW release. Once activated, RfaH hyper-stabilizes the pause, which thus requires anti-backtracking factors for escape. Our results suggest that the entire RfaH cycle is solely determined by the ops and RfaH sequences and provide insights into mechanisms of recruitment and metamorphosis of NusG homologs across all life.
History
DepositionJun 22, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17685.png

Downloads & links

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Map

FileDownload / File: emd_17685.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 319.488 Å		0.83 Å/pix.
x 384 pix.
= 319.488 Å		0.83 Å/pix.
x 384 pix.
= 319.488 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.0 - 1.5737714
Average (Standard dev.)0.010134836 (±0.05291628)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 319.488 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_17685_half_map_1.map
Projections & Slices
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Density Histograms

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Half map: #2

Fileemd_17685_half_map_2.map
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Sample components

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Entire : transcription complex paused at ops site, assembled on a fully co...

EntireName: transcription complex paused at ops site, assembled on a fully complementary nucleic acid scaffold, bound to RfaH and NusA
Components
  • Complex: transcription complex paused at ops site, assembled on a fully complementary nucleic acid scaffold, bound to RfaH and NusA
    • Complex: Transcription complex
      • Protein or peptide: Transcription antitermination protein RfaH
      • Protein or peptide: DNA-directed RNA polymerase subunit betaPolymerase
      • Protein or peptide: DNA-directed RNA polymerase subunit beta'Polymerase
      • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
      • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
      • Protein or peptide: Transcription termination/antitermination protein NusA
    • Complex: DNA and RNA
      • DNA: non-template DNA
      • DNA: template DNA
      • RNA: RNA
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: transcription complex paused at ops site, assembled on a fully co...

SupramoleculeName: transcription complex paused at ops site, assembled on a fully complementary nucleic acid scaffold, bound to RfaH and NusA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Molecular weightTheoretical: 472 KDa

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Supramolecule #2: Transcription complex

SupramoleculeName: Transcription complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#5, #9
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: DNA and RNA

SupramoleculeName: DNA and RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #6-#8
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Transcription antitermination protein RfaH

MacromoleculeName: Transcription antitermination protein RfaH / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 18.559414 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GHMQSWYLLY CKRGQLQRAQ EHLERQAVNC LAPMITLEKI VRGKRTAVSE PLFPNYLFVE FDPEVIHTTT INATRGVSHF VRFGASPAI VPSAVIHQLS VYKPKDIVDP ATPYPGDKVI ITEGAFEGFQ AIFTEPDGEA RSMLLLNLIN KEIKHSVKNT E FRKL

UniProtKB: Transcription antitermination protein RfaH

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Macromolecule #2: DNA-directed RNA polymerase subunit beta

MacromoleculeName: DNA-directed RNA polymerase subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
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 #3: DNA-directed RNA polymerase subunit beta'

MacromoleculeName: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 156.504969 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: VKDLLKFLKA QTKTEEFDAI KIALASPDMI RSWSFGEVKK PETINYRTFK PERDGLFCAR IFGPVKDYEC LCGKYKRLKH RGVICEKCG VEVTQTKVRR ERMGHIELAS PTAHIWFLKS LPSRIGLLLD MPLRDIERVL YFESYVVIEG GMTNLERQQI L TEEQYLDA ...String:
VKDLLKFLKA 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 SDNELEVHHH HHH

UniProtKB: DNA-directed RNA polymerase subunit beta'

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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 (E. coli)
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: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
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 #9: Transcription termination/antitermination protein NusA

MacromoleculeName: Transcription termination/antitermination protein NusA
type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 55.086848 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPMNKEILAV VEAVSNEKAL PREKIFEALE SALATATKKK YEQEIDVRVQ IDRKSGDFDT FRRWLVVDEV TQPTKEITLE AARYEDESL NLGDYVEDQI ESVTFDRITT QTAKQVIVQK VREAERAMVV DQFREHEGEI ITGVVKKVNR DNISLDLGNN A EAVILRED ...String:
GPMNKEILAV VEAVSNEKAL PREKIFEALE SALATATKKK YEQEIDVRVQ IDRKSGDFDT FRRWLVVDEV TQPTKEITLE AARYEDESL NLGDYVEDQI ESVTFDRITT QTAKQVIVQK VREAERAMVV DQFREHEGEI ITGVVKKVNR DNISLDLGNN A EAVILRED MLPRENFRPG DRVRGVLYSV RPEARGAQLF VTRSKPEMLI ELFRIEVPEI GEEVIEIKAA ARDPGSRAKI AV KTNDKRI DPVGACVGMR GARVQAVSTE LGGERIDIVL WDDNPAQFVI NAMAPADVAS IVVDEDKHTM DIAVEAGNLA QAI GRNGQN VRLASQLSGW ELNVMTVDDL QAKHQAEAHA AIDTFTKYLD IDEDFATVLV EEGFSTLEEL AYVPMKELLE IEGL DEPTV EALRERAKNA LATIAQAQEE SLGDNKPADD LLNLEGVDRD LAFKLAARGV CTLEDLAEQG IDDLADIEGL TDEKA GALI MAARNICWFG DEA

UniProtKB: Transcription termination/antitermination protein NusA

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Macromolecule #6: non-template DNA

MacromoleculeName: non-template DNA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 12.207802 KDa
SequenceString:
(DC)(DA)(DC)(DC)(DA)(DC)(DC)(DA)(DC)(DG) (DC)(DG)(DG)(DG)(DC)(DG)(DG)(DT)(DA)(DG) (DC)(DG)(DT)(DG)(DC)(DT)(DT)(DT)(DT) (DT)(DT)(DC)(DG)(DA)(DT)(DC)(DT)(DT)(DC) (DC)

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Macromolecule #7: template DNA

MacromoleculeName: template DNA / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 12.421979 KDa
SequenceString:
(DG)(DG)(DA)(DA)(DG)(DA)(DT)(DC)(DG)(DA) (DA)(DA)(DA)(DA)(DA)(DG)(DC)(DA)(DC)(DG) (DC)(DT)(DA)(DC)(DC)(DG)(DC)(DC)(DC) (DG)(DC)(DG)(DT)(DG)(DG)(DT)(DG)(DG)(DT) (DG)

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Macromolecule #8: RNA

MacromoleculeName: RNA / type: rna / ID: 8 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 5.414189 KDa
SequenceString:
UUCUUUGGCG GUAGCGU

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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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Macromolecule #11: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 11 / 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

Concentration3.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMTris
120.0 mMKOAc
5.0 mMMg(OAc)2
0.01 mMZnCl2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3142 / Average exposure time: 40.57 sec. / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 877027
Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.2.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 41797
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 121
Output model

PDB-8pil:
E. coli transcription complex paused at ops site and bound to RfaH and NusA

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