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- EMDB-34476: Cryo-EM structure of the transcription activation complex NtcA-TAC -

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Basic information

Entry
Database: EMDB / ID: EMD-34476
TitleCryo-EM structure of the transcription activation complex NtcA-TAC
Map data
Sample
  • Complex: transcription activation complex with the global regulator NtcA
    • DNA: DNA (125-MER)
    • DNA: DNA (125-MER)
    • 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 alphaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit gammaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
    • Protein or peptide: RNA polymerase sigma factor SigA
    • Protein or peptide: NtcA
KeywordsTranscription activation complex / TRANSCRIPTION
Function / homology
Function and homology information


sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-binding transcription factor activity / DNA-templated transcription / magnesium ion binding ...sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-binding transcription factor activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
RNA polymerase sigma factor, RpoD-like, cyanobacteria / Transcription regulator, NtcA / DNA-directed RNA polymerase, subunit gamma / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / 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 ...RNA polymerase sigma factor, RpoD-like, cyanobacteria / Transcription regulator, NtcA / DNA-directed RNA polymerase, subunit gamma / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / 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 / 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 / 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, 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, 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 Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / 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, beta subunit, protrusion / RNA polymerase beta subunit / 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 / 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
Global nitrogen regulator / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit gamma / DNA-directed RNA polymerase subunit beta' / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesAnabaena (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHan SJ / Jiang YL / You LL / Shen LQ / Wu XX / Yang F / Kong WW / Chen ZP / Zhang Y / Zhou CZ
Funding support China, 4 items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB37020301, XDA24020302 China
National Natural Science Foundation of China (NSFC)32171198 China
Ministry of Science and Technology (MoST, China)2018YFA090070 China
Chinese Academy of Sciences2020452 China
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: DNA looping mediates cooperative transcription activation.
Authors: Shu-Jing Han / Yong-Liang Jiang / Lin-Lin You / Li-Qiang Shen / Xiaoxian Wu / Feng Yang / Ning Cui / Wen-Wen Kong / Hui Sun / Ke Zhou / Hui-Chao Meng / Zhi-Peng Chen / Yuxing Chen / Yu Zhang / Cong-Zhao Zhou /
Abstract: Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here ...Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here we report two cryo-electron microscopy structures of Anabaena transcription activation complexes (TACs): NtcA-TAC composed of RNAP holoenzyme, promoter and a global activator NtcA, and NtcA-NtcB-TAC comprising an extra context-specific regulator, NtcB. Structural analysis showed that NtcA binding makes the promoter DNA bend by ∼50°, which facilitates RNAP to contact NtcB at the distal upstream NtcB box. The sequential binding of NtcA and NtcB induces looping back of promoter DNA towards RNAP, enabling the assembly of a fully activated TAC bound with two activators. Together with biochemical assays, we propose a 'DNA looping' mechanism of cooperative transcription activation in bacteria.
History
DepositionOct 9, 2022-
Header (metadata) releaseOct 4, 2023-
Map releaseOct 4, 2023-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_34476.png

Downloads & links

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Map

FileDownload / File: emd_34476.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.0149228405 - 0.045231134
Average (Standard dev.)0.00010415183 (±0.002013405)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34476_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34476_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : transcription activation complex with the global regulator NtcA

EntireName: transcription activation complex with the global regulator NtcA
Components
  • Complex: transcription activation complex with the global regulator NtcA
    • DNA: DNA (125-MER)
    • DNA: DNA (125-MER)
    • 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 alphaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit gammaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
    • Protein or peptide: RNA polymerase sigma factor SigA
    • Protein or peptide: NtcA

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Supramolecule #1: transcription activation complex with the global regulator NtcA

SupramoleculeName: transcription activation complex with the global regulator NtcA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Anabaena (bacteria) / Strain: PCC. 7120
Molecular weightTheoretical: 580 KDa

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Macromolecule #1: DNA (125-MER)

MacromoleculeName: DNA (125-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Anabaena (bacteria)
Molecular weightTheoretical: 38.723902 KDa
SequenceString: (DG)(DT)(DT)(DA)(DA)(DG)(DT)(DG)(DT)(DA) (DA)(DT)(DG)(DC)(DA)(DA)(DA)(DA)(DA)(DA) (DC)(DG)(DC)(DA)(DT)(DA)(DT)(DT)(DC) (DT)(DC)(DT)(DA)(DT)(DG)(DC)(DA)(DA)(DA) (DA) (DA)(DA)(DC)(DG)(DC)(DA) ...String:
(DG)(DT)(DT)(DA)(DA)(DG)(DT)(DG)(DT)(DA) (DA)(DT)(DG)(DC)(DA)(DA)(DA)(DA)(DA)(DA) (DC)(DG)(DC)(DA)(DT)(DA)(DT)(DT)(DC) (DT)(DC)(DT)(DA)(DT)(DG)(DC)(DA)(DA)(DA) (DA) (DA)(DA)(DC)(DG)(DC)(DA)(DT)(DT) (DA)(DA)(DT)(DA)(DC)(DG)(DA)(DG)(DA)(DA) (DT)(DT) (DT)(DT)(DG)(DT)(DA)(DG)(DC) (DT)(DA)(DC)(DT)(DT)(DA)(DT)(DA)(DC)(DA) (DA)(DA)(DA) (DT)(DT)(DC)(DA)(DG)(DG) (DA)(DA)(DA)(DA)(DT)(DT)(DT)(DT)(DT)(DC) (DT)(DG)(DT)(DA) (DT)(DA)(DA)(DT)(DG) (DG)(DG)(DA)(DG)(DC)(DT)(DG)(DT)(DC)(DA) (DC)(DG)(DG)(DA)(DT) (DG)(DC)(DA)(DG) (DG)

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Macromolecule #2: DNA (125-MER)

MacromoleculeName: DNA (125-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Anabaena (bacteria)
Molecular weightTheoretical: 38.571723 KDa
SequenceString: (DC)(DC)(DT)(DG)(DC)(DA)(DT)(DC)(DC)(DG) (DT)(DG)(DA)(DG)(DT)(DC)(DG)(DA)(DG)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DA)(DC)(DA) (DG)(DA)(DA)(DA)(DA)(DA)(DT)(DT)(DT)(DT) (DC) (DC)(DT)(DG)(DA)(DA)(DT) ...String:
(DC)(DC)(DT)(DG)(DC)(DA)(DT)(DC)(DC)(DG) (DT)(DG)(DA)(DG)(DT)(DC)(DG)(DA)(DG)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DA)(DC)(DA) (DG)(DA)(DA)(DA)(DA)(DA)(DT)(DT)(DT)(DT) (DC) (DC)(DT)(DG)(DA)(DA)(DT)(DT)(DT) (DT)(DG)(DT)(DA)(DT)(DA)(DA)(DG)(DT)(DA) (DG)(DC) (DT)(DA)(DC)(DA)(DA)(DA)(DA) (DT)(DT)(DC)(DT)(DC)(DG)(DT)(DA)(DT)(DT) (DA)(DA)(DT) (DG)(DC)(DG)(DT)(DT)(DT) (DT)(DT)(DT)(DG)(DC)(DA)(DT)(DA)(DG)(DA) (DG)(DA)(DA)(DT) (DA)(DT)(DG)(DC)(DG) (DT)(DT)(DT)(DT)(DT)(DT)(DG)(DC)(DA)(DT) (DT)(DA)(DC)(DA)(DC) (DT)(DT)(DA)(DA) (DC)

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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: Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576
Molecular weightTheoretical: 126.781531 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVSSKKFNQV DKGRGMISEN YIEPAFLLPD LIEIQRSSFR WFLEEGLIEE LNSFSPITDY TGKLELHFLG HNYKLKEPKY SVEEAKRRD STYAVQMYVP TRLLNKETGD IKEQEVFIGD LPLMTDRGTF IINGAERVIV NQIVRSPGVY YKSEIDKNGR R TYSASLIP ...String:
MVSSKKFNQV DKGRGMISEN YIEPAFLLPD LIEIQRSSFR WFLEEGLIEE LNSFSPITDY TGKLELHFLG HNYKLKEPKY SVEEAKRRD STYAVQMYVP TRLLNKETGD IKEQEVFIGD LPLMTDRGTF IINGAERVIV NQIVRSPGVY YKSEIDKNGR R TYSASLIP NRGAWLKFET DRNDLVWVRI DKTRKLSAQV LLKALGLSDN EIFDALRHPE YFQKTIEKEG QFSEEEALME LY RKLRPGE PPTVLGGQQL LDSRFFDPKR YDLGRVGRYK LNKKLRLSVP DTVRVLTSGD ILAAVDYLIN LEYDIGSIDD IDH LGNRRV RSVGELLQNQ VRVGLNRLER IIRERMTVSD AEVLTPASLV NPKPLVAAIK EFFGSSQLSQ FMDQTNPLAE LTHK RRLSA LGPGGLTRER AGFAVRDIHP SHYGRICPIE TPEGPNAGLI GSLATHARVN QYGFLETPFR PVENGRVRFD QPAAY MTAD EEDDLRVAPG DIPVDENGYI IGPQVPVRYR QEFSTTTPEQ VDYVAVSPVQ IVSVATSMIP FLEHDDANRA LMGSNM QRQ AVPLLKPERP LVGTGLEAQG ARDSGMVIVS RTDGDVVYVD ATEIRVRVSG QLPTASGKST DNGQLTSQKG QEIRYTV SK YQRSNQDTCL NQKPLVRIGE RVVAGQVLAD GSSTEGGELA LGQNIVVAYM PWEGYNYEDA ILISERLVQD DIYTSIHI E KYEIEARQTK LGPEEITREI PNVGEDALRQ LDEQGIIRIG AWVEAGDILV GKVTPKGESD QPPEEKLLRA IFGEKARDV RDNSLRVPNG EKGRVVDVRL FTREQGDELP PGANMVVRVY VAQKRKIQVG DKMAGRHGNK GIISRILPIE DMPYLPDGSP VDIVLNPLG VPSRMNVGQV FECLLGWAGH TLGVRFKITP FDEMYGEESS RRIVHGKLQE ARDETGKDWV YNPDDPGKIM V YDGRTGEA FDRPVTIGVA YMLKLVHLVD DKIHARSTGP YSLVTQQPLG GKAQQGGQRF GEMEVWALEA FGAAYTLQEL LT VKSDDMQ GRNEALNAIV KGKAIPRPGT PESFKVLMRE LQSLGLDIAV HKVETQADGS SLDVEVDLMA DQLARRTPPR PTY ESLSRE SLDDDE

UniProtKB: DNA-directed RNA polymerase subunit beta

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Macromolecule #4: DNA-directed RNA polymerase subunit beta'

MacromoleculeName: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576
Molecular weightTheoretical: 147.137484 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIFRNRVVDK GQLRNLISWA FTHYGTARTA VMADKLKDLG FRYATRAGVS ISVDDLMVPP SKRSLLEAAE EEIRATEVRY QRGEITEVE RFQKVIDTWN GTSEALKDEV VTHFKQTNPL NSVYMMAFSG ARGNISQVRQ LVGMRGLMAD PQGEIIDLPI K TNFREGLT ...String:
MIFRNRVVDK GQLRNLISWA FTHYGTARTA VMADKLKDLG FRYATRAGVS ISVDDLMVPP SKRSLLEAAE EEIRATEVRY QRGEITEVE RFQKVIDTWN GTSEALKDEV VTHFKQTNPL NSVYMMAFSG ARGNISQVRQ LVGMRGLMAD PQGEIIDLPI K TNFREGLT VTEYIISSYG ARKGLVDTAL RTADSGYLTR RLVDVSQDVI IREIDCGTTR GIPVRPMTEG SKTLIKLSTR LL GRVVGED VIHPKTKEVI APRNTPISDD LAKEIEKAGV AEVVVRSPLT CEAARSVCQH CYGWSLAHAK MVDLGEAVGI IAA QSIGEP GTQLTMRTFH TGGVFTGEVA QQVRSKMDGT IKLPRKLRTR THRTRHGEDA LFVESNGIMI LEPRKEGSET PAPQ EIHVT QGSTIYIVDG QQVKKGQLLA EVALGGRTTR TNTEKAVKDV ASDLAGEVKF AEVVPEQKTD RQGNTTTTAA RGGLI WILS GEVYNLPPGA ELVVKNGDRV ETNGVLAETK LTTIHGGVVR LPEATPGKST REIEIITASV VLDQATVTVQ SSQGRN NYL ITTGNNQVFN LRATPGTKVQ NGQVVAELID DRYRTTTGGF LKFAGVEVQK KGKAKLGYEV VQGGTLLWIP EETHEVN KD ISLLLVEDGQ YVEAGTEVVK DIFCQNSGVV EVTQKNDILR EVVVKPGELL MVDDPEAVIG RDNTLLQPGE ELLGQVAT E LRYIQYVESP EGPALLSRPV VEFAVPSNPD VPSTTSVSQQ TGRSIQMRAV QRLPYKDSER VKSVEGVELL RTQLVLEIE QEGEQEHNAS PLAADIELIP DLEDADVQRL QLVILESLVL RRDIAADATQ GSTQTSLEVK DGDTIVPGSV VARTQILSKE GGIVRGVQK GSEAVRRCLV LRHSDMATLN ISAKPKVKAG DLIVAGTELA PGIFAEESGQ IVGVKNAGES TTTQDAALST Q NYAVTIRA GRPYRVSPGA VLQIEDGDLV QRGDNLVLLV FERAKTGDII QGLPRIEELL EARKPKEACI LAKRGGEVKV VY GDGDEAI AIKVIESNGV VTDYPLGPGQ NLAMPDGSVV PAGQPLSDGP SNPHEILEVF FSLGSEDGVY ACASHALQKV QTF LVNEVQ MVYQSQGIDI ADKHIEVIVR QMTNKVRIDD GGDTTMLPGE LVELRQVEQV NEAMAITGGA RAQYTPVLLG ITKA SLNTD SFISAASFQE TTRVLTEAAI EGKSDWLRGL KENVIIGRLI PAGTGYNTYD EPGMLEDYST LETTSVLDET DDPLD MVLD DRTARAYNLD SPGLAETGFN NRRSILDDDE LIADEIHDLV EAEVEVDDEV DDDYEDDDED DDDYED

UniProtKB: DNA-directed RNA polymerase subunit beta'

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Macromolecule #5: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576
Molecular weightTheoretical: 26.166613 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVAQFQIECV ESNTEESRNH YSKFILEPLE RGQGTTVGNA LRRVLLSNLE GTAVTAVRIA GVSHEFATVP GVREDVLEII MRMKEVILK SYSSQAQIGR LLVNGPTTIT ASHFDLPSEV EVIDPTQYVA TIAEGGKLEM EFRIERGKGY RTVERGREEA T SLDFLQID ...String:
MVAQFQIECV ESNTEESRNH YSKFILEPLE RGQGTTVGNA LRRVLLSNLE GTAVTAVRIA GVSHEFATVP GVREDVLEII MRMKEVILK SYSSQAQIGR LLVNGPTTIT ASHFDLPSEV EVIDPTQYVA TIAEGGKLEM EFRIERGKGY RTVERGREEA T SLDFLQID SIFMPVRKVN YSVEEVRADG SIPKDRLLLE VWTNGSISPQ EALSSAAGIL VDLFNPLKDI SLEPTDTN

UniProtKB: DNA-directed RNA polymerase subunit alpha

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Macromolecule #6: DNA-directed RNA polymerase subunit gamma

MacromoleculeName: DNA-directed RNA polymerase subunit gamma / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576
Molecular weightTheoretical: 70.653055 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRPAQTNQFD YVKIGLASPE RIRQWGERTL PNGQVVGEVT KPETINYRTL KPEMDGLFCE RIFGPAKDWE CHCGKYKRVR HRGIVCERC GVEVTESRVR RHRMGYIKLA APVAHVWYLK GIPSYISILL DMPLRDVEQI VYFNSYVVLS PGNAETLTYK Q LLSEDQWL ...String:
MRPAQTNQFD YVKIGLASPE RIRQWGERTL PNGQVVGEVT KPETINYRTL KPEMDGLFCE RIFGPAKDWE CHCGKYKRVR HRGIVCERC GVEVTESRVR RHRMGYIKLA APVAHVWYLK GIPSYISILL DMPLRDVEQI VYFNSYVVLS PGNAETLTYK Q LLSEDQWL EIEDQIYSED SQLQGVEVGI GAEALLRLLA DINLEQEAES LREEIGSAKG QKRAKLIKRL RVIDNFIATG SK PEWMVMT VIPVIPPDLR PMVQLDGGRF ATSDLNDLYR RVINRNNRLA RLQEILAPEI IVRNEKRMLQ EAVDALIDNG RRG RTVVGA NNRPLKSLSD IIEGKQGRFR QNLLGKRVDY SGRSVIVVGP KLKIHQCGLP REMAIELFQP FVINRLIRSG MVNN IKAAK KLISRNDPSV WDVLEEVIEG HPVMLNRAPT LHRLGIQAFE PILVEGRAIQ LHPLVCPAFN ADFDGDQMAV HVPLS LESQ AEARLLMLAS NNILSPATGR PIITPSQDMV LGAYYLTAEN PGATKGAGKY FASLDDVIMA FQQEQIDLHA YVYVRF DGD VESDQPDTEP VKVTTNEDGS RTVLYKYRRV REDAQGNVIS QYIYTTPGRV IYNKAIQEAL AS

UniProtKB: DNA-directed RNA polymerase subunit gamma

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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: Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576
Molecular weightTheoretical: 9.14349 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MLKRSKFETT QSQIMHRAED LISAASNRYR ITVQVANRAK RRRYEEFESA EDAMMKPVLR AIIEMSDELT QPEIIGEI

UniProtKB: DNA-directed RNA polymerase subunit omega

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Macromolecule #8: RNA polymerase sigma factor SigA

MacromoleculeName: RNA polymerase sigma factor SigA / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576
Molecular weightTheoretical: 45.717027 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNQANNVLDS IYQPDLEIMN QPEIELDDLL IEEDEDLLLA DDGDIDEFLE PQTDEDDAKS GKAAKSRRRT QSKKKHYTED SIRLYLQEI GRIRLLRADE EIELARKIAD LLELERVRER LSEKLERDPR DSEWAEAVQL PLPAFRYRLH IGRRAKDKMV Q SNLRLVVS ...String:
MNQANNVLDS IYQPDLEIMN QPEIELDDLL IEEDEDLLLA DDGDIDEFLE PQTDEDDAKS GKAAKSRRRT QSKKKHYTED SIRLYLQEI GRIRLLRADE EIELARKIAD LLELERVRER LSEKLERDPR DSEWAEAVQL PLPAFRYRLH IGRRAKDKMV Q SNLRLVVS IAKKYMNRGL SFQDLIQEGS LGLIRAAEKF DHEKGYKFST YATWWIRQAI TRAIADQSRT IRLPVHLYET IS RIKKTTK LLSQEMGRKP TEEEIATRME MTIEKLRFIA KSAQLPISLE TPIGKEEDSR LGDFIESDGE TPEDQVSKNL LRE DLEKVL DSLSPRERDV LRLRYGLDDG RMKTLEEIGQ IFNVTRERIR QIEAKALRKL RHPNRNSVLK EYIR

UniProtKB: RNA polymerase sigma factor SigA

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Macromolecule #9: NtcA

MacromoleculeName: NtcA / type: protein_or_peptide / ID: 9 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576
Molecular weightTheoretical: 24.951266 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIVTQDKALA NVFRQMATGA FPPVVETFER NKTIFFPGDP AERVYFLLKG AVKLSRVYEA GEEITVALLR ENSVFGVLSL LTGNKSDRF YHAVAFTPVE LLSAPIEQVE QALKENPELS MLMLRGLSSR ILQTEMMIET LAHRDMGSRL VSFLLILCRD F GVPCADGI ...String:
MIVTQDKALA NVFRQMATGA FPPVVETFER NKTIFFPGDP AERVYFLLKG AVKLSRVYEA GEEITVALLR ENSVFGVLSL LTGNKSDRF YHAVAFTPVE LLSAPIEQVE QALKENPELS MLMLRGLSSR ILQTEMMIET LAHRDMGSRL VSFLLILCRD F GVPCADGI TIDLKLSHQA IAEAIGSTRV TVTRLLGDLR EKKMISIHKK KITVHKPVTL SRQFT

UniProtKB: Global nitrogen regulator

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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: 7.5
Component:
ConcentrationFormulaName
100.0 mMKClpotassium chloride
5.0 mMMgCl2magnesium chloride
10.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
2.0 mMDTTDithiothreitol

Details: 10 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2 and 2 mM DTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 1462 / Average electron dose: 50.0 e/Å2
Details: Images were collected in movie-mode at 32 frames per second.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 555921
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8gzg


Details: 8GZG and 3LA2 are required for the reconstruction of Nt cA-TAC
Initial angle assignmentType: PROJECTION MATCHING
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 45239
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8h40:
Cryo-EM structure of the transcription activation complex NtcA-TAC

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