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- EMDB-41439: Escherichia coli RNA polymerase unwinding intermediate (I1b) at t... -

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Basic information

Entry
Database: EMDB / ID: EMD-41439
TitleEscherichia coli RNA polymerase unwinding intermediate (I1b) at the lambda PR promoter
Map datamelting intermediate I1b map filtered by local resolution
Sample
  • Complex: Escherichia coli RNA polymerase unwinding intermediate (I1b) at the lambda PR promoter
    • 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'
    • Protein or peptide: DNA-directed RNA polymerase subunit omega
    • Protein or peptide: RNA polymerase sigma factor RpoD
    • DNA: Nontemplate strand of lamdba PR promoter DNA
    • DNA: Template strand of lamdba PR promoter DNA
  • Ligand: (3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-10,13-dimethyl-17-[(2R)-pentan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-triol
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
KeywordsDNA-dependent RNA polymerase / transcription / intermediate / DNA promoter / DNA unwinding / transcription-DNA complex
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 / 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 (E. coli) / Escherichia coli (strain K12) (bacteria) / Escherichia coli K-12 (bacteria) / Escherichia phage Lambda (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsDarst SA / Saecker RM / Mueller AU
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118130 United States
Citation
Journal: Nat Struct Mol Biol / Year: 2024
Title: Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.
Authors: Ruth M Saecker / Andreas U Mueller / Brandon Malone / James Chen / William C Budell / Venkata P Dandey / Kashyap Maruthi / Joshua H Mendez / Nina Molina / Edward T Eng / Laura Y Yen / ...Authors: Ruth M Saecker / Andreas U Mueller / Brandon Malone / James Chen / William C Budell / Venkata P Dandey / Kashyap Maruthi / Joshua H Mendez / Nina Molina / Edward T Eng / Laura Y Yen / Clinton S Potter / Bridget Carragher / Seth A Darst /
Abstract: During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural ...During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ-RNAP and the λP promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.
#1: Journal: Nat.Struct.Mol.Biol. / Year: 2024
Title: Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy
Authors: Darst SA / Saecker RM / Mueller AU / Malone B / Chen J / Budell WC / Dandey VP / Maruthi K / Mendez JH / Molina N / Eng ET / Yen LY / Potter CS / Carragher B
History
DepositionAug 3, 2023-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41439.png

Downloads & links

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Map

FileDownload / File: emd_41439.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmelting intermediate I1b map filtered by local resolution
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 384 pix.
= 324.096 Å		0.84 Å/pix.
x 384 pix.
= 324.096 Å		0.84 Å/pix.
x 384 pix.
= 324.096 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.844 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-2.7325158 - 3.3726132
Average (Standard dev.)0.0028232795 (±0.069088)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 324.096 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: melting intermediate I1b unsharpened map

Fileemd_41439_additional_1.map
Annotationmelting intermediate I1b unsharpened map
Projections & Slices
AxesZYX

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Additional map: melting intermediate I1b sharpened map

Fileemd_41439_additional_2.map
Annotationmelting intermediate I1b sharpened map
Projections & Slices
AxesZYX

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Half map: melting intermediate I1b half map A

Fileemd_41439_half_map_1.map
Annotationmelting intermediate I1b half map A
Projections & Slices
AxesZYX

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Density Histograms

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Histogram (log scale)

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Half map: melting intermediate I1b half map B

Fileemd_41439_half_map_2.map
Annotationmelting intermediate I1b half map B
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Sample components

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Entire : Escherichia coli RNA polymerase unwinding intermediate (I1b) at t...

EntireName: Escherichia coli RNA polymerase unwinding intermediate (I1b) at the lambda PR promoter
Components
  • Complex: Escherichia coli RNA polymerase unwinding intermediate (I1b) at the lambda PR promoter
    • 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'
    • Protein or peptide: DNA-directed RNA polymerase subunit omega
    • Protein or peptide: RNA polymerase sigma factor RpoD
    • DNA: Nontemplate strand of lamdba PR promoter DNA
    • DNA: Template strand of lamdba PR promoter DNA
  • Ligand: (3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-10,13-dimethyl-17-[(2R)-pentan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-triol
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Escherichia coli RNA polymerase unwinding intermediate (I1b) at t...

SupramoleculeName: Escherichia coli RNA polymerase unwinding intermediate (I1b) at the lambda PR promoter
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (strain K12) (bacteria)
Molecular weightTheoretical: 36.55868 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (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 #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 (strain K12) (bacteria)
Molecular weightTheoretical: 150.820875 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (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 (strain K12) (bacteria)
Molecular weightTheoretical: 155.366781 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (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 SDNE

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 (strain K12) (bacteria)
Molecular weightTheoretical: 10.249547 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (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)
Molecular weightTheoretical: 70.352242 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (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 GIDMNTDYTL EEVGKQFDVT RERIRQIEAK ALRKLRHPSR SEVLRSFLDD

UniProtKB: RNA polymerase sigma factor RpoD

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Macromolecule #6: Nontemplate strand of lamdba PR promoter DNA

MacromoleculeName: Nontemplate strand of lamdba PR promoter DNA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia phage Lambda (virus)
Molecular weightTheoretical: 32.600814 KDa
SequenceString: (DC)(DG)(DG)(DA)(DA)(DT)(DC)(DG)(DA)(DG) (DG)(DG)(DA)(DT)(DC)(DC)(DT)(DC)(DT)(DA) (DG)(DA)(DG)(DT)(DT)(DG)(DG)(DA)(DT) (DA)(DA)(DA)(DT)(DA)(DT)(DC)(DT)(DA)(DA) (DC) (DA)(DC)(DC)(DG)(DT)(DG) ...String:
(DC)(DG)(DG)(DA)(DA)(DT)(DC)(DG)(DA)(DG) (DG)(DG)(DA)(DT)(DC)(DC)(DT)(DC)(DT)(DA) (DG)(DA)(DG)(DT)(DT)(DG)(DG)(DA)(DT) (DA)(DA)(DA)(DT)(DA)(DT)(DC)(DT)(DA)(DA) (DC) (DA)(DC)(DC)(DG)(DT)(DG)(DC)(DG) (DT)(DG)(DT)(DT)(DG)(DA)(DC)(DT)(DA)(DT) (DT)(DT) (DT)(DA)(DC)(DC)(DT)(DC)(DT) (DG)(DG)(DC)(DG)(DG)(DT)(DG)(DA)(DT)(DA) (DA)(DT)(DG) (DG)(DT)(DT)(DG)(DC)(DA) (DT)(DG)(DT)(DA)(DC)(DT)(DA)(DA)(DG)(DG) (DA)(DG)(DG)(DT) (DT)(DG)(DT)(DC)(DG)

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Macromolecule #7: Template strand of lamdba PR promoter DNA

MacromoleculeName: Template strand of lamdba PR promoter DNA / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia phage Lambda (virus)
Molecular weightTheoretical: 32.183631 KDa
SequenceString: (DC)(DG)(DA)(DC)(DA)(DA)(DC)(DC)(DT)(DC) (DC)(DT)(DT)(DA)(DG)(DT)(DA)(DC)(DA)(DT) (DG)(DC)(DA)(DA)(DC)(DC)(DA)(DT)(DT) (DA)(DT)(DC)(DA)(DC)(DC)(DG)(DC)(DC)(DA) (DG) (DA)(DG)(DG)(DT)(DA)(DA) ...String:
(DC)(DG)(DA)(DC)(DA)(DA)(DC)(DC)(DT)(DC) (DC)(DT)(DT)(DA)(DG)(DT)(DA)(DC)(DA)(DT) (DG)(DC)(DA)(DA)(DC)(DC)(DA)(DT)(DT) (DA)(DT)(DC)(DA)(DC)(DC)(DG)(DC)(DC)(DA) (DG) (DA)(DG)(DG)(DT)(DA)(DA)(DA)(DA) (DT)(DA)(DG)(DT)(DC)(DA)(DA)(DC)(DA)(DC) (DG)(DC) (DA)(DC)(DG)(DG)(DT)(DG)(DT) (DT)(DA)(DG)(DA)(DT)(DA)(DT)(DT)(DT)(DA) (DT)(DC)(DC) (DA)(DA)(DC)(DT)(DC)(DT) (DA)(DG)(DA)(DG)(DG)(DA)(DT)(DC)(DC)(DC) (DT)(DC)(DG)(DA) (DT)(DT)(DC)(DC)(DG)

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Macromolecule #8: (3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-10,13-dimethyl-17-[(2R)-pent...

MacromoleculeName: (3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-10,13-dimethyl-17-[(2R)-pentan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-triol
type: ligand / ID: 8 / Number of copies: 4 / Formula: 4QM
Molecular weightTheoretical: 378.588 Da
Chemical component information

ChemComp-4QM:
(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-10,13-dimethyl-17-[(2R)-pentan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-triol

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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

BufferpH: 8
Component:
ConcentrationFormulaName
40.0 mMTris
120.0 mMKClpotassium chloride
10.0 mMMgCl2magnesium chloride
10.0 mMDTT1,4-dithiothreitol
VitrificationCryogen name: ETHANE / Chamber temperature: 296 K / Instrument: SPOTITON
Details: CHAPSO was added (from 80 mM stock) to 8 mM final in each sample just prior to spray mixing..
Detailstip 1: 26 or 30 micromolar Es70 RNAP tip 2: 52 or 60 micromolar LPR DNA

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsPlease see publication for details.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Details: Cryo-EM map from images from multiple data collections, please see publication.
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio generated map
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: NU refinement / Number images used: 153655
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.2.0) / Software - details: 3DVA

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