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- EMDB-41856: Cryo-EM structure of E. coli NarL-transcription activation comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-41856
TitleCryo-EM structure of E. coli NarL-transcription activation complex at 3.2A
Map dataE coli NarL-TAC unsharpened map
Sample
  • Complex: E. coli NarL transcription activation complex on the yeaR promoter
    • Protein or peptide: x 6 types
    • DNA: x 2 types
    • RNA: x 1 types
  • Ligand: x 2 types
Keywordstranscription / RNA polymerase / transcription activation / NarL / nitrate-responsive regulator / transcription-DNA complex
Function / homology
Function and homology information


sigma factor antagonist complex / DNA-binding transcription repressor activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / phosphorelay signal transduction system ...sigma factor antagonist complex / DNA-binding transcription repressor activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / phosphorelay signal transduction system / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation / DNA-templated transcription initiation / protein-DNA complex / transcription antitermination / cell motility / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / transcription cis-regulatory region binding / protein dimerization activity / response to antibiotic / negative regulation of DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / ATP binding / identical protein binding / membrane / cytosol / cytoplasm
Similarity search - Function
LuxR-type HTH domain signature. / LuxR-type HTH domain profile. / Transcription regulator LuxR, C-terminal / Bacterial regulatory proteins, luxR family / helix_turn_helix, Lux Regulon / Transcriptional regulatory protein WalR-like / 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 ...LuxR-type HTH domain signature. / LuxR-type HTH domain profile. / Transcription regulator LuxR, C-terminal / Bacterial regulatory proteins, luxR family / helix_turn_helix, Lux Regulon / Transcriptional regulatory protein WalR-like / 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 / Signal transduction response regulator, C-terminal effector / 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 / Response regulator receiver domain / cheY-homologous receiver domain / Signal transduction response regulator, receiver domain / Response regulatory domain profile. / CheY-like 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 / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / 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, 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 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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-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 / Nitrate/nitrite response regulator protein NarL
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsLiu B / Kompaniiets D / Wang D
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Structural basis for transcription activation by the nitrate-responsive regulator NarL.
Authors: Dmytro Kompaniiets / Lina He / Dong Wang / Wei Zhou / Yang Yang / Yangbo Hu / Bin Liu /
Abstract: Transcription activation is a crucial step of regulation during transcription initiation and a classic check point in response to different stimuli and stress factors. The Escherichia coli NarL is a ...Transcription activation is a crucial step of regulation during transcription initiation and a classic check point in response to different stimuli and stress factors. The Escherichia coli NarL is a nitrate-responsive global transcription factor that controls the expression of nearly 100 genes. However, the molecular mechanism of NarL-mediated transcription activation is not well defined. Here we present a cryo-EM structure of NarL-dependent transcription activation complex (TAC) assembled on the yeaR promoter at 3.2 Å resolution. Our structure shows that the NarL dimer binds at the -43.5 site of the promoter DNA with its C-terminal domain (CTD) not only binding to the DNA but also making interactions with RNA polymerase subunit alpha CTD (αCTD). The key role of these NarL-mediated interactions in transcription activation was further confirmed by in vivo and in vitro transcription assays. Additionally, the NarL dimer binds DNA in a different plane from that observed in the structure of class II TACs. Unlike the canonical class II activation mechanism, NarL does not interact with σ4, while RNAP αCTD is bound to DNA on the opposite side of NarL. Our findings provide a structural basis for detailed mechanistic understanding of NarL-dependent transcription activation on yeaR promoter and reveal a potentially novel mechanism of transcription activation.
History
DepositionSep 7, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateFeb 21, 2024-
Current statusFeb 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41856.png

Downloads & links

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Map

FileDownload / File: emd_41856.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE coli NarL-TAC unsharpened map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.89 Å/pix.
x 384 pix.
= 339.968 Å		0.89 Å/pix.
x 384 pix.
= 339.968 Å		0.89 Å/pix.
x 384 pix.
= 339.968 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.88533 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.35114324 - 0.7116764
Average (Standard dev.)0.0013024393 (±0.020329671)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 339.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: E coli NarL-TAC sharpened map

Fileemd_41856_additional_1.map
AnnotationE coli NarL-TAC sharpened map
Projections & Slices
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Additional map: Local refinement map on the NarL-DNA region

Fileemd_41856_additional_2.map
AnnotationLocal refinement map on the NarL-DNA region
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Half map: E coli NarL-TAC half map A

Fileemd_41856_half_map_1.map
AnnotationE coli NarL-TAC half map A
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Half map: E coli NarL-TAC half map B

Fileemd_41856_half_map_2.map
AnnotationE coli NarL-TAC half map B
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Sample components

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Entire : E. coli NarL transcription activation complex on the yeaR promoter

EntireName: E. coli NarL transcription activation complex on the yeaR promoter
Components
  • Complex: E. coli NarL transcription activation complex on the yeaR 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
    • Protein or peptide: Nitrate/nitrite response regulator protein NarL
    • DNA: DNA (69-MER)
    • DNA: DNA (69-MER)
    • RNA: RNA (5'-D(*(GTP))-R(P*AP*A)-3')
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: E. coli NarL transcription activation complex on the yeaR promoter

SupramoleculeName: E. coli NarL transcription activation complex on the yeaR promoter
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 625 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 (E. coli)
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 (E. coli)
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 (E. coli)
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 (E. coli)
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 (E. coli)
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: Nitrate/nitrite response regulator protein NarL

MacromoleculeName: Nitrate/nitrite response regulator protein NarL / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 8.903552 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
RDVNQLTPRE RDILKLIAQG LPNKMIARRL DITESTVKVH VKHMLKKMKL KSRVEAAVWV HQERIFLEHH HHHH

UniProtKB: Nitrate/nitrite response regulator protein NarL

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Macromolecule #7: DNA (69-MER)

MacromoleculeName: DNA (69-MER) / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 21.307693 KDa
SequenceString: (DA)(DG)(DT)(DA)(DA)(DC)(DC)(DA)(DA)(DT) (DA)(DA)(DA)(DT)(DG)(DG)(DT)(DA)(DT)(DT) (DT)(DA)(DA)(DA)(DA)(DT)(DG)(DC)(DA) (DA)(DA)(DT)(DT)(DA)(DT)(DC)(DA)(DG)(DG) (DC) (DG)(DT)(DA)(DC)(DC)(DC) ...String:
(DA)(DG)(DT)(DA)(DA)(DC)(DC)(DA)(DA)(DT) (DA)(DA)(DA)(DT)(DG)(DG)(DT)(DA)(DT)(DT) (DT)(DA)(DA)(DA)(DA)(DT)(DG)(DC)(DA) (DA)(DA)(DT)(DT)(DA)(DT)(DC)(DA)(DG)(DG) (DC) (DG)(DT)(DA)(DC)(DC)(DC)(DT)(DC) (DT)(DT)(DT)(DG)(DC)(DG)(DA)(DA)(DT)(DT) (DC)(DG) (DC)(DG)(DG)(DC)(DA)(DG)(DC) (DG)(DG)

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Macromolecule #8: DNA (69-MER)

MacromoleculeName: DNA (69-MER) / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 21.164541 KDa
SequenceString: (DC)(DC)(DG)(DC)(DT)(DG)(DC)(DC)(DG)(DC) (DG)(DA)(DA)(DT)(DT)(DC)(DC)(DG)(DT)(DT) (DT)(DC)(DA)(DG)(DG)(DG)(DT)(DA)(DC) (DG)(DC)(DC)(DT)(DG)(DA)(DT)(DA)(DA)(DT) (DT) (DT)(DG)(DC)(DA)(DT)(DT) ...String:
(DC)(DC)(DG)(DC)(DT)(DG)(DC)(DC)(DG)(DC) (DG)(DA)(DA)(DT)(DT)(DC)(DC)(DG)(DT)(DT) (DT)(DC)(DA)(DG)(DG)(DG)(DT)(DA)(DC) (DG)(DC)(DC)(DT)(DG)(DA)(DT)(DA)(DA)(DT) (DT) (DT)(DG)(DC)(DA)(DT)(DT)(DT)(DT) (DA)(DA)(DA)(DT)(DA)(DC)(DC)(DA)(DT)(DT) (DT)(DA) (DT)(DT)(DG)(DG)(DT)(DT)(DA) (DC)(DT)

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Macromolecule #9: RNA (5'-D(*(GTP))-R(P*AP*A)-3')

MacromoleculeName: RNA (5'-D(*(GTP))-R(P*AP*A)-3') / type: rna / ID: 9 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 1.118619 KDa
SequenceString:
(GTP)AA

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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

Concentration15 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 290 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5769 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 8727 / Average exposure time: 2.0 sec. / Average electron dose: 42.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2554259
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 104500
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8u3b:
Cryo-EM structure of E. coli NarL-transcription activation complex at 3.2A

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