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- EMDB-33468: cryo-EM structure of HK022 putRNA-less E.coli RNA polymerase elon... -

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Basic information

Entry
Database: EMDB / ID: EMD-33468
Titlecryo-EM structure of HK022 putRNA-less E.coli RNA polymerase elongation complex
Map datalocal-filtered map after post-process
Sample
  • Complex: HK022 putRNA-less E.coli RNA polymerase elongation complex
    • Complex: E.coli RNA polymerase
      • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
      • 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
    • Complex: DNA/RNA
      • DNA: non-template DNA
      • DNA: template DNA
      • RNA: RNA (nun gene and immunity region)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


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 / regulation of DNA-templated transcription elongation ...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 / regulation of DNA-templated transcription elongation / transcription antitermination / cell motility / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
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 / RNA polymerase Rpb6 ...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 / 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
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
Similarity search - Component
Biological speciesEscherichia coli (strain K12) (bacteria) / Shamshuipovirus HK022
Methodsingle particle reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsHwang S / Kang JY
Funding support Korea, Republic Of, United States, 5 items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)NRF-2019R1F1A1064026 Korea, Republic Of
National Research Foundation (NRF, Korea)NRF-2019M3E5D6066058 Korea, Republic Of
National Research Foundation (NRF, Korea)NRF-2021R1C1C100656011 Korea, Republic Of
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM109824 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41 GM103314 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis of transcriptional regulation by a nascent RNA element, HK022 putRNA.
Authors: Seungha Hwang / Paul Dominic B Olinares / Jimin Lee / Jinwoo Kim / Brian T Chait / Rodney A King / Jin Young Kang /
Abstract: Transcription, in which RNA polymerases (RNAPs) produce RNA from DNA, is the first step of gene expression. As such, it is highly regulated either by trans-elements like protein factors and/or by cis- ...Transcription, in which RNA polymerases (RNAPs) produce RNA from DNA, is the first step of gene expression. As such, it is highly regulated either by trans-elements like protein factors and/or by cis-elements like specific sequences on the DNA. Lambdoid phage HK022 contains a cis-element, put, which suppresses pausing and termination during transcription of the early phage genes. The putRNA transcript solely performs the anti-pausing/termination activities by interacting directly with the E.coli RNAP elongation complex (EC) by an unknown structural mechanism. In this study, we reconstituted putRNA-associated ECs and determined the structures using cryo-electron microscopy. The determined structures of putRNA-associated EC, putRNA-absent EC, and σ-bound EC suggest that the putRNA interaction with the EC counteracts swiveling, a conformational change previously identified to promote pausing and σ might modulate putRNA folding via σ-dependent pausing during elongation.
History
DepositionMay 18, 2022-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateAug 31, 2022-
Current statusAug 31, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33468.png

Downloads & links

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Map

FileDownload / File: emd_33468.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal-filtered map after post-process
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.07806769 - 0.1167621
Average (Standard dev.)0.00012893918 (±0.002312019)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions296296296
Spacing296296296
CellA=B=C: 313.75998 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : HK022 putRNA-less E.coli RNA polymerase elongation complex

EntireName: HK022 putRNA-less E.coli RNA polymerase elongation complex
Components
  • Complex: HK022 putRNA-less E.coli RNA polymerase elongation complex
    • Complex: E.coli RNA polymerase
      • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
      • 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
    • Complex: DNA/RNA
      • DNA: non-template DNA
      • DNA: template DNA
      • RNA: RNA (nun gene and immunity region)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: HK022 putRNA-less E.coli RNA polymerase elongation complex

SupramoleculeName: HK022 putRNA-less E.coli RNA polymerase elongation complex
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Molecular weightTheoretical: 440 KDa

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Supramolecule #2: E.coli RNA polymerase

SupramoleculeName: E.coli RNA polymerase / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #4-#7
Source (natural)Organism: Escherichia coli (strain K12) (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: DNA/RNA

SupramoleculeName: DNA/RNA / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #1-#3

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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: Shamshuipovirus HK022
Molecular weightTheoretical: 54.756973 KDa
SequenceString: (DG)(DC)(DA)(DT)(DG)(DA)(DA)(DT)(DT)(DC) (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) ...String:
(DG)(DC)(DA)(DT)(DG)(DA)(DA)(DT)(DT)(DC) (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)(DG)(DC)(DG)(DT)(DC)(DT)(DT)(DT)(DA) (DA)(DA)(DC) (DC)(DA)(DT)(DG)(DC)(DG) (DT)(DC)(DG)(DG)(DG)(DA)(DG)(DC)(DG)(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)(DC)(DT)(DT)(DT)(DT) (DA)(DG)(DA)(DA)(DT)(DT)(DG) (DT)(DG) (DA)(DG)(DC)(DG)(DC)(DT)(DC)(DA)(DC)(DA) (DA)(DT)(DT)(DC)(DG)

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Macromolecule #2: template DNA

MacromoleculeName: template DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Shamshuipovirus HK022
Molecular weightTheoretical: 54.512855 KDa
SequenceString: (DC)(DG)(DA)(DA)(DT)(DT)(DG)(DT)(DG)(DA) (DG)(DC)(DG)(DC)(DT)(DC)(DA)(DC)(DA)(DA) (DT)(DT)(DC)(DT)(DA)(DA)(DA)(DA)(DG) (DC)(DA)(DA)(DA)(DA)(DA)(DA)(DG)(DC)(DC) (DT) (DT)(DC)(DT)(DC)(DG)(DC) ...String:
(DC)(DG)(DA)(DA)(DT)(DT)(DG)(DT)(DG)(DA) (DG)(DC)(DG)(DC)(DT)(DC)(DA)(DC)(DA)(DA) (DT)(DT)(DC)(DT)(DA)(DA)(DA)(DA)(DG) (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)(DC)(DG)(DC)(DT) (DC)(DC)(DC)(DG)(DA)(DC)(DG)(DC)(DA)(DT) (DG)(DG)(DT)(DT) (DT)(DA)(DA)(DA)(DG) (DA)(DC)(DG)(DC)(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)(DG)(DA)(DA)(DT)(DT) (DC)(DA)(DT)(DG)(DC)

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Macromolecule #3: RNA (nun gene and immunity region)

MacromoleculeName: RNA (nun gene and immunity region) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Shamshuipovirus HK022
Molecular weightTheoretical: 30.714211 KDa
SequenceString:
AUAGACGAAC GGCGCGUCUU UAAACCAUGC GUCGGGAGCG CGGCGGGUUC AGGAUGAACG GCAAUGCUGC UCAUUAGCGA GAAGGCUUU UUUGCU

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Macromolecule #4: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 4 / Number of copies: 2 / 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 (E. coli)
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

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Macromolecule #5: DNA-directed RNA polymerase subunit beta

MacromoleculeName: DNA-directed RNA polymerase subunit beta / type: protein_or_peptide / ID: 5 / 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 (E. coli)
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

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Macromolecule #6: DNA-directed RNA polymerase subunit beta'

MacromoleculeName: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (strain K12) (bacteria)
Molecular weightTheoretical: 158.073609 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 SDNELELEVL FQGPSSGHHH HHHHHHH

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Macromolecule #7: DNA-directed RNA polymerase subunit omega

MacromoleculeName: DNA-directed RNA polymerase subunit omega / type: protein_or_peptide / ID: 7 / 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 (E. coli)
SequenceString:
MARVTVQDAV EKIGNRFDLV LVAARRARQM QVGGKDPLVP EENDKTTVIA LREIEEGLIN NQILDVRERQ EQQEQEAAEL QAVTAIAEG RR

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3trisaminomethane
10.0 mMMg(C5H8NO4)2magnesium glutamate
150.0 mMKC5H8NO4potassium glutamate
5.0 mMC4H10O2S2Dithiothreitol

Details: pH adjustment at 4'C
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: using two layers of blot papers.

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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: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8175 / Average electron dose: 42.16 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 411900
CTF correctionSoftware - Name: Gctf
Startup modelType of model: EMDB MAP
EMDB ID:

8585

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Avg.num./class: 137000 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 103900

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Atomic model buiding 1

Initial modelPDB ID:

6c6t

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7xug:
cryo-EM structure of HK022 putRNA-less E.coli RNA polymerase elongation complex

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Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

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