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- EMDB-7059: The cryo-EM structure of a bacterial class I transcription activa... -

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Basic information

Entry
Database: EMDB / ID: EMD-7059
TitleThe cryo-EM structure of a bacterial class I transcription activation complex
Map dataClass I transcription activation complex
Sample
  • Organelle or cellular component: The complex of class-I bacterial transcription activation complex
    • Protein or peptide: x 7 types
    • DNA: x 2 types
    • RNA: x 1 types
  • Ligand: x 3 types
Keywordstranscription / RNA polymerase / catabolite activator protein / cAMP / TRANSCRIPTION-TRANSFERASE-DNA-RNA complex
Function / homology
Function and homology information


sigma factor antagonist complex / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / cAMP binding / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase ...sigma factor antagonist complex / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / cAMP binding / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein dimerization activity / DNA-binding transcription factor activity / negative regulation of DNA-templated transcription / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / helix_turn_helix, cAMP Regulatory protein / Crp-type HTH domain profile. / Crp-like helix-turn-helix domain / Crp-type HTH domain / 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 ...Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / helix_turn_helix, cAMP Regulatory protein / Crp-type HTH domain profile. / Crp-like helix-turn-helix domain / Crp-type HTH domain / 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 / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Sigma-70 factors family signature 1. / Cyclic nucleotide-binding, conserved site / 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 / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / 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 / RmlC-like jelly roll fold / 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 / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / cAMP-activated global transcriptional regulator CRP
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli O157:H7 (bacteria) / Escherichia coli O45:K1 (strain S88 / ExPEC) (bacteria) / Escherichia coli O45:K1 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsLiu B / Hong C
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM22778 United States
CitationJournal: Science / Year: 2017
Title: Structural basis of bacterial transcription activation.
Authors: Bin Liu / Chuan Hong / Rick K Huang / Zhiheng Yu / Thomas A Steitz /
Abstract: In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic ...In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact class I TAC containing a CAP dimer, a σ-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.
History
DepositionOct 2, 2017-
Header (metadata) releaseNov 1, 2017-
Map releaseNov 15, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.13
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6b6h
  • Surface level: 0.13
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7059.png

Downloads & links

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Map

FileDownload / File: emd_7059.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClass I transcription activation complex
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.13 / Movie #1: 0.13
Minimum - Maximum-0.079181686 - 0.36147064
Average (Standard dev.)0.004876344 (±0.022157894)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0790.3610.005

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Supplemental data

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Sample components

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Entire : The complex of class-I bacterial transcription activation complex

EntireName: The complex of class-I bacterial transcription activation complex
Components
  • Organelle or cellular component: The complex of class-I bacterial transcription activation complex
    • 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
    • Protein or peptide: RNA polymerase sigma factor RpoD
    • Protein or peptide: cAMP-activated global transcriptional regulator CRP
    • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
    • DNA: SYNTHETIC NONTEMPLATE STRAND DNA (88-MER)
    • DNA: SYNTHETIC TEMPLATE STRAND DNA (88-MER)
    • RNA: NASCENT RNA 3-MER
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE

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Supramolecule #1: The complex of class-I bacterial transcription activation complex

SupramoleculeName: The complex of class-I bacterial transcription activation complex
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
Details: A CAP dimer, a sigma70-RNA polymerase holoenzyme, an intact CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 590 KDa

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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: 2 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli O157:H7 (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

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 O45:K1 (strain S88 / ExPEC) (bacteria)
Strain: S88 / ExPEC
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

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 O157:H7 (bacteria)
Molecular weightTheoretical: 155.366781 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 O45:K1 (bacteria) / Strain: S88 / ExPEC
Molecular weightTheoretical: 10.249547 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 72.206266 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRGSHHHHHH TDQFTMEQNP QSQLKLLVTR GKEQGYLTYA EVNDHLPEDI VDSDQIEDII QMINDMGIQV MEEAPDADDL MLAENTADE DAAEAAAQVL SSVESEIGRT TDPVRMYMRE MGTVELLTRE GEIDIAKRIE DGINQVQCSV AEYPEAITYL L EQYDRVEA ...String:
MRGSHHHHHH TDQFTMEQNP QSQLKLLVTR GKEQGYLTYA EVNDHLPEDI VDSDQIEDII QMINDMGIQV MEEAPDADDL MLAENTADE DAAEAAAQVL SSVESEIGRT TDPVRMYMRE MGTVELLTRE GEIDIAKRIE DGINQVQCSV AEYPEAITYL L EQYDRVEA EEARLSDLIT GFVDPNAEED LAPTATHVGS ELSQEDLDDD EDEDEEDGDD DSADDDNSID PELAREKFAE LR AQYVVTR DTIKAKGRSH ATAQEEILKL SEVFKQFRLV PKQFDYLVNS MRVMMDRVRT QERLIMKLCV EQCKMPKKNF ITL FTGNET SDTWFNAAIA MNKPWSEKLH DVSEEVHRAL QKLQQIEEET GLTIEQVKDI NRRMSIGEAK ARRAKKEMVE ANLR LVISI AKKYTNRGLQ FLDLIQEGNI GLMKAVDKFE YRRGYKFSTY ATWWIRQAIT RSIADQARTI RIPVHMIETI NKLNR ISRQ MLQEMGREPT PEELAERMLM PEDKIRKVLK IAKEPISMET PIGDDEDSHL GDFIEDTTLE LPLDSATTES LRAATH DVL AGLTAREAKV LRMRFGIDMN TDYTLEEVGK QFDVTRERIR QIEAKALRKL RHPSRSEVLR SFLDD

UniProtKB: RNA polymerase sigma factor RpoD

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Macromolecule #6: cAMP-activated global transcriptional regulator CRP

MacromoleculeName: cAMP-activated global transcriptional regulator CRP / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli O157:H7 (bacteria)
Molecular weightTheoretical: 23.672439 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVLGKPQTDP TLEWFLSHCH IHKYPSKSTL IHQGEKAETL YYIVKGSVAV LIKDEEGKEM ILSYLNQGDF IGELGLFEEG QERSAWVRA KTACEVAEIS YKKFRQLIQV NPDILMRLSA QMARRLQVTS EKVGNLAFLD VTGRIAQTLL NLAKQPDAMT H PDGMQIKI ...String:
MVLGKPQTDP TLEWFLSHCH IHKYPSKSTL IHQGEKAETL YYIVKGSVAV LIKDEEGKEM ILSYLNQGDF IGELGLFEEG QERSAWVRA KTACEVAEIS YKKFRQLIQV NPDILMRLSA QMARRLQVTS EKVGNLAFLD VTGRIAQTLL NLAKQPDAMT H PDGMQIKI TRQEIGQIVG CSRETVGRIL KMLEDQNLIS AHGKTIVVYG TR

UniProtKB: cAMP-activated global transcriptional regulator CRP

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Macromolecule #7: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli O157:H7 (bacteria)
Molecular weightTheoretical: 8.346699 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
DPILLRPVDD LELTVRSANC LKAEAIHYIG DLVQRTEVEL LKTPNLGKKS LTEIKDVLAS RGLSLGMRLE NWPPA

UniProtKB: DNA-directed RNA polymerase subunit alpha

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Macromolecule #8: SYNTHETIC NONTEMPLATE STRAND DNA (88-MER)

MacromoleculeName: SYNTHETIC NONTEMPLATE STRAND DNA (88-MER) / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 27.133393 KDa
SequenceString: (DT)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DG)(DA) (DT)(DC)(DT)(DA)(DG)(DA)(DT)(DC)(DA)(DC) (DA)(DT)(DT)(DT)(DT)(DA)(DG)(DG)(DC) (DA)(DA)(DA)(DA)(DA)(DA)(DG)(DG)(DC)(DC) (DT) (DT)(DG)(DA)(DC)(DA)(DT) ...String:
(DT)(DA)(DA)(DA)(DA)(DT)(DG)(DT)(DG)(DA) (DT)(DC)(DT)(DA)(DG)(DA)(DT)(DC)(DA)(DC) (DA)(DT)(DT)(DT)(DT)(DA)(DG)(DG)(DC) (DA)(DA)(DA)(DA)(DA)(DA)(DG)(DG)(DC)(DC) (DT) (DT)(DG)(DA)(DC)(DA)(DT)(DC)(DC) (DC)(DA)(DC)(DC)(DT)(DC)(DA)(DC)(DG)(DT) (DA)(DT) (DG)(DC)(DT)(DA)(DT)(DA)(DA) (DT)(DG)(DT)(DG)(DT)(DG)(DC)(DA)(DG)(DT) (DC)(DT)(DG) (DA)(DC)(DG)(DC)(DG)(DG) (DC)(DG)

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Macromolecule #9: SYNTHETIC TEMPLATE STRAND DNA (88-MER)

MacromoleculeName: SYNTHETIC TEMPLATE STRAND DNA (88-MER) / type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 27.217404 KDa
SequenceString: (DC)(DG)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DA) (DG)(DA)(DC)(DT)(DC)(DG)(DT)(DA)(DG)(DG) (DA)(DT)(DT)(DA)(DT)(DA)(DG)(DC)(DA) (DT)(DA)(DC)(DG)(DT)(DG)(DA)(DG)(DG)(DT) (DG) (DG)(DG)(DA)(DT)(DG)(DT) ...String:
(DC)(DG)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DA) (DG)(DA)(DC)(DT)(DC)(DG)(DT)(DA)(DG)(DG) (DA)(DT)(DT)(DA)(DT)(DA)(DG)(DC)(DA) (DT)(DA)(DC)(DG)(DT)(DG)(DA)(DG)(DG)(DT) (DG) (DG)(DG)(DA)(DT)(DG)(DT)(DC)(DA) (DA)(DG)(DG)(DC)(DC)(DT)(DT)(DT)(DT)(DT) (DT)(DG) (DC)(DC)(DT)(DA)(DA)(DA)(DA) (DT)(DG)(DT)(DG)(DA)(DT)(DC)(DT)(DA)(DG) (DA)(DT)(DC) (DA)(DC)(DA)(DT)(DT)(DT) (DT)(DA)

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Macromolecule #10: NASCENT RNA 3-MER

MacromoleculeName: NASCENT RNA 3-MER / type: rna / ID: 10 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 1.134619 KDa
SequenceString:
(GTP)AG

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Macromolecule #11: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 11 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #12: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 12 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #13: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE

MacromoleculeName: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / type: ligand / ID: 13 / Number of copies: 2 / Formula: CMP
Molecular weightTheoretical: 329.206 Da
Chemical component information

ChemComp-CMP:
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / Cyclic adenosine monophosphate

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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 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMTris
5.0 mMMgCl2
0.1 mMEDTAEthylenediaminetetraacetic acid
50.0 mMNaClSodium chloride
5.0 mMDTT

Details: 20 mM TRIS pH 7.5, 50 mM sodium chloride, 0.1mM EDTA, 5 mM MgCl2, 5 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 3 second blotting.

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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 / Calibrated defocus max: 2.6 µm / Calibrated defocus min: 1.2 µm / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 80.0 K
DetailsCs corrector
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 2382 / Average exposure time: 0.25 sec. / Average electron dose: 1.37 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 835000
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4ipl

Initial angle assignmentType: OTHER / Software - Name: RELION
Final 3D classificationNumber classes: 4 / Software - Name: RELION
Final angle assignmentType: OTHER / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 161000
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient
Output model

PDB-6b6h:
The cryo-EM structure of a bacterial class I transcription activation complex

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