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- EMDB-35047: Cryo-EM structure of AfsR-dependent transcription activation comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-35047
TitleCryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter
Map data
Sample
  • Complex: Cryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter
    • Protein or peptide: x 8 types
    • DNA: x 2 types
  • Ligand: x 2 types
KeywordsRNA polymerase / SARP regulator / gene regulation / TRANSCRIPTION
Function / homology
Function and homology information


pigment binding / bacterial-type RNA polymerase holo enzyme binding / phosphorelay signal transduction system / sigma factor activity / rRNA transcription / antibiotic biosynthetic process / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / ADP binding ...pigment binding / bacterial-type RNA polymerase holo enzyme binding / phosphorelay signal transduction system / sigma factor activity / rRNA transcription / antibiotic biosynthetic process / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / ADP binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / ATP binding / cytoplasm
Similarity search - Function
Bacterial transcriptional activator domain / Bacterial transcriptional activator domain / Bacterial transcriptional activator domain / : / CarD, C-terminal domain / CarD-like, C-terminal domain / CarD-like/TRCF, RNAP-interacting domain / CarD-like/TRCF, RNAP-interacting domain superfamily / CarD-like/TRCF RID domain / CarD-like/TRCF domain ...Bacterial transcriptional activator domain / Bacterial transcriptional activator domain / Bacterial transcriptional activator domain / : / CarD, C-terminal domain / CarD-like, C-terminal domain / CarD-like/TRCF, RNAP-interacting domain / CarD-like/TRCF, RNAP-interacting domain superfamily / CarD-like/TRCF RID domain / CarD-like/TRCF domain / Tetratricopeptide repeat / NB-ARC / NB-ARC domain / OmpR/PhoB-type DNA-binding domain profile. / OmpR/PhoB-type DNA-binding domain / Transcriptional regulatory protein, C terminal / Transcriptional regulatory protein, C terminal / Tetratricopeptide repeat / 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 / 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 / TPR repeat region circular profile. / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / 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 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 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 / 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 / Tetratricopeptide-like helical domain superfamily / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit alpha / : / RNA polymerase principal sigma factor HrdB / Regulatory protein AfsR / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / Transcriptional factor regulator
Similarity search - Component
Biological speciesStreptomyces coelicolor A3(2) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsWang Y / Zheng J
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31770068, 32070040 China
CitationJournal: PLoS Biol / Year: 2024
Title: Structural and functional characterization of AfsR, an SARP family transcriptional activator of antibiotic biosynthesis in Streptomyces.
Authors: Yiqun Wang / Xu Yang / Feng Yu / Zixin Deng / Shuangjun Lin / Jianting Zheng /
Abstract: Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide- ...Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the -35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β' zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.
History
DepositionDec 27, 2022-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateApr 3, 2024-
Current statusApr 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35047.png

Downloads & links

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Map

FileDownload / File: emd_35047.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.055
Minimum - Maximum-1.0877182 - 1.9156817
Average (Standard dev.)0.0020112633 (±0.033106994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 377.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35047_msk_1.map
Projections & Slices
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Half map: #2

Fileemd_35047_half_map_1.map
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Half map: #1

Fileemd_35047_half_map_2.map
Projections & Slices
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Sample components

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Entire : Cryo-EM structure of AfsR-dependent transcription activation comp...

EntireName: Cryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter
Components
  • Complex: Cryo-EM structure of AfsR-dependent transcription activation complex with afsS 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 principal sigma factor HrdB
    • Protein or peptide: Putative transcriptional factor regulator
    • Protein or peptide: Regulatory protein AfsR
    • DNA: DNA (65-MER)
    • DNA: DNA (65-MER)
    • Protein or peptide: RNA polymerase-binding protein RbpA
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Cryo-EM structure of AfsR-dependent transcription activation comp...

SupramoleculeName: Cryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5, #7-#10, #6
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 560 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: 3 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 36.734641 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLIAQRPSLT EEVVDEFRSR FVIEPLEPGF GYTLGNSLRR TLLSSIPGAA VTSIRIDGVL HEFTTVPGVK EDVTDLILNI KQLVVSSEH DEPVVMYLRK QGPGLVTAAD IAPPAGVEVH NPDLVLATLN GKGKLEMELT VERGRGYVSA VQNKQVGQEI G RIPVDSIY ...String:
MLIAQRPSLT EEVVDEFRSR FVIEPLEPGF GYTLGNSLRR TLLSSIPGAA VTSIRIDGVL HEFTTVPGVK EDVTDLILNI KQLVVSSEH DEPVVMYLRK QGPGLVTAAD IAPPAGVEVH NPDLVLATLN GKGKLEMELT VERGRGYVSA VQNKQVGQEI G RIPVDSIY SPVLKVTYKV EATRVEQRTD FDKLIVDVET KQAMRPRDAM ASAGKTLVEL FGLARELNID AEGIDMGPSP TD AALAADL ALPIEELELT VRSYNCLKRE GIHSVGELVA RSEADLLDIR NFGAKSIDEV KAKLAGMGLA LKDSPPGFDP TAA ADAFGA DDDADAGFVE TEQY

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: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 128.644945 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAASRNASTA NTNNAASTAP LRISFAKIKE PLEVPNLLAL QTESFDWLLG NDAWKARVES ALESGQDVPT KSGLEEIFEE ISPIEDFSG SMSLTFRDHR FEPPKNSIDE CKDRDFTYAA PLFVTAEFTN NETGEIKSQT VFMGDFPLMT NKGTFVINGT E RVVVSQLV ...String:
MAASRNASTA NTNNAASTAP LRISFAKIKE PLEVPNLLAL QTESFDWLLG NDAWKARVES ALESGQDVPT KSGLEEIFEE ISPIEDFSG SMSLTFRDHR FEPPKNSIDE CKDRDFTYAA PLFVTAEFTN NETGEIKSQT VFMGDFPLMT NKGTFVINGT E RVVVSQLV RSPGVYFDSS IDKTSDKDIF SAKIIPSRGA WLEMEIDKRD MVGVRIDRKR KQSVTVLLKA LGWTTEQILE EF GEYESMR ATLEKDHTQG QDDALLDIYR KLRPGEPPTR EAAQTLLENL YFNPKRYDLA KVGRYKVNKK LGADEPLDAG VLT TDDVIA TIKYLVKLHA GETETVGESG REIVVETDDI DHFGNRRIRN VGELIQNQVR TGLARMERVV RERMTTQDVE AITP QTLIN IRPVVASIKE FFGTSQLSQF MDQNNPLSGL THKRRLNALG PGGLSRERAG FEVRDVHPSH YGRMCPIETP EGPNI GLIG SLASYGRINP FGFIETPYRK VVEGQVTDDV DYLTADEEDR FVIAQANAAL GDDMRFAEAR VLVRRRGGEV DYVPGD DVD YMDVSPRQMV SVATAMIPFL EHDDANRALM GANMMRQAVP LIKSESPLVG TGMEYRSAAD AGDVVKAEKA GVVQEVS AD YITTTNDDGT YITYRLAKFS RSNQGTSVNQ KVIVAEGDRI IEGQVLADGP ATENGEMALG KNLLVAFMPW EGHNYEDA I ILSQRLVQDD VLSSIHIEEH EVDARDTKLG PEEITRDIPN VSEEVLADLD ERGIIRIGAE VVAGDILVGK VTPKGETEL TPEERLLRAI FGEKAREVRD TSLKVPHGEI GKVIGVRVFD REEGDELPPG VNQLVRVYVA QKRKITDGDK LAGRHGNKGV ISKINPIED MPFLEDGTPV DIILNPLAVP SRMNPGQVLE IHLGWLASRG WDVSGLAEEW AQRLQVIGAD KVEPGTNVAT P VFDGARED ELAGLLQHTI PNRDGERMVL PSGKARLFDG RSGEPFPEPI SVGYMYILKL HHLVDDKLHA RSTGPYSMIT QQ PLGGKAQ FGGQRFGEME VWALEAYGAA YALQELLTIK SDDVTGRVKV YEAIVKGENI PEPGIPESFK VLIKEMQSLC LNV EVLSSD GMSIEMRDTD EDVFRAAEEL GIDLSRREPS SVEEV

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: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 145.912219 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLDVNFFDEL RIGLATADDI RQWSHGEVKK PETINYRTLK PEKDGLFCEK IFGPTRDWEC YCGKYKRVRF KGIICERCGV EVTRAKVRR ERMGHIELAA PVTHIWYFKG VPSRLGYLLD LAPKDLEKVI YFAAYMITFV DEERRTRDLP SLEAHVSVER Q QIEQRRDS ...String:
MLDVNFFDEL RIGLATADDI RQWSHGEVKK PETINYRTLK PEKDGLFCEK IFGPTRDWEC YCGKYKRVRF KGIICERCGV EVTRAKVRR ERMGHIELAA PVTHIWYFKG VPSRLGYLLD LAPKDLEKVI YFAAYMITFV DEERRTRDLP SLEAHVSVER Q QIEQRRDS DLEARAKKLE TDLAELEAEG AKADVRRKVR EGAEREMKQL RDRAQREIDR LDEVWNRFKN LKVQDLEGDE LL YRELRDR FGTYFDGSMG AAALQKRLES FDLDEEAERL REIIRTGKGQ KKTRALKRLK VVSAFLQTSN SPKGMVLDCV PVI PPDLRP MVQLDGGRFA TSDLNDLYRR VINRNNRLKR LLDLGAPEII VNNEKRMLQE AVDALFDNGR RGRPVTGPGN RPLK SLSDM LKGKQGRFRQ NLLGKRVDYS ARSVIVVGPQ LKLHQCGLPK AMALELFKPF VMKRLVDLNH AQNIKSAKRM VERGR TVVY DVLEEVIAEH PVLLNRAPTL HRLGIQAFEP QLVEGKAIQI HPLVCTAFNA DFDGDQMAVH LPLSAEAQAE ARILML SSN NILKPADGRP VTMPTQDMVL GLFFLTTDSE GRSPKGEGRA FGSSAEAIMA FDAGDLTLQA KIDIRFPVGT IPPRGFE PP AREEGEPEWQ QGDTFTLKTT LGRALFNELL PEDYPFVDYE VGKKQLSEIV NDLAERYPKV IVAATLDNLK AAGFFWAT R SGVTVAISDI VVPDAKKEIV KGYEGQDEKV QKQYERGLIT KEERTQELIA IWTKATNEVA EAMNDNFPKT NPVSMMVNS GARGNMMQMR QIAGMRGLVS NAKNETIPRP IKASFREGLS VLEYFISTHG ARKGLADTAL RTADSGYLTR RLVDVSQDVI IREEDCGTE RGLKLPIATR DADGTLRKAE DVETSVYARM LAEDVVIDGK VIAPANVDLG DVLIDALVAH GVEEVKTRSI L TCESQVGT CAMCYGRSLA TGKLVDIGEA VGIIAAQSIG EPGTQLTMRT FHTGGVAGDD ITQGLPRVVE LFEARTPKGV AP ISEASGR VRIEETEKTK KIVVTPDDGS DETAFPISKR ARLLVGEGDH VEVGQKLTVG ATNPHDVLRI LGQRAVQVHL VGE VQKVYN SQGVSIHDKH IEIIIRQMLR RVTIIESGDA ELLPGELVER TKFETENRRV VQEGGHPASG RPQLMGITKA SLAT ESWLS AASFQETTRV LTDAAINAKS DSLIGLKENV IIGKLIPAGT GLSRYRNIRV EPTEEAKAAM YSAVGYDDID YSPFG TGSG QAVPLEDYDY GPYNQHHHHH HHH

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: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 9.716941 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSSSISAPEG IINPPIDELL EATDSKYSLV IYAAKRARQI NAYYSQLGEG LLEYVGPLVD THVHEKPLSI ALREINAGLL TSEAIEGPA Q

UniProtKB: DNA-directed RNA polymerase subunit omega

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Macromolecule #5: RNA polymerase principal sigma factor HrdB

MacromoleculeName: RNA polymerase principal sigma factor HrdB / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 58.188953 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MSASTSRTLP PEIAESVSVM ALIERGKAEG QIAGDDVRRA FEADQIPATQ WKNVLRSLNQ ILEEEGVTL MVSAAEPKRT RKSVAAKSPA KRTATKAVAA KPVTSRKATA PAAPAAPATE PAAVEEEAPA KKAAAKKTTA K KATAKKTT ...String:
MGSSHHHHHH SSGLVPRGSH MSASTSRTLP PEIAESVSVM ALIERGKAEG QIAGDDVRRA FEADQIPATQ WKNVLRSLNQ ILEEEGVTL MVSAAEPKRT RKSVAAKSPA KRTATKAVAA KPVTSRKATA PAAPAAPATE PAAVEEEAPA KKAAAKKTTA K KATAKKTT AKKAAAKKTT AKKEDGELLE DEATEEPKAA TEEPEGTENA GFVLSDEDED DAPAQQVAAA GATADPVKDY LK QIGKVPL LNAEQEVELA KRIEAGLFAE DKLANSDKLA PKLKRELEII AEDGRRAKNH LLEANLRLVV SLAKRYTGRG MLF LDLIQE GNLGLIRAVE KFDYTKGYKF STYATWWIRQ AITRAMADQA RTIRIPVHMV EVINKLARVQ RQMLQDLGRE PTPE ELAKE LDMTPEKVIE VQKYGREPIS LHTPLGEDGD SEFGDLIEDS EAVVPADAVS FTLLQEQLHS VLDTLSEREA GVVSM RFGL TDGQPKTLDE IGKVYGVTRE RIRQIESKTM SKLRHPSRSQ VLRDYLD

UniProtKB: RNA polymerase principal sigma factor HrdB

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Macromolecule #6: RNA polymerase-binding protein RbpA

MacromoleculeName: RNA polymerase-binding protein RbpA / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 14.146094 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSERALRGTR LVVTSYETDR GIDLAPRQAV EYACEKGHRF EMPFSVEAEI PPEWECKVCG AQALLVDGDG PEEKKAKPAR THWDMLMER RTREELEEVL EERLAVLRSG AMNIAVHPRD SRKSA

UniProtKB: UNIPROTKB: A0A6M9XI42

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Macromolecule #7: Putative transcriptional factor regulator

MacromoleculeName: Putative transcriptional factor regulator / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 17.855338 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MTFKVGDTVV YPHHGAALIE AIETRQIKGV DKTYLVLKVA QGDLTVRVPA DNAEFVGVRD VVGQDGLDRV FEVLRAPYAE EPTNWSRRY KANLEKLASG DVIKVAEVVR DLWRRERERG LSAGEKRMLA KARQILVSEL ALAENTNEDK AEALLDEVLA S

UniProtKB: Transcriptional factor regulator

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Macromolecule #8: Regulatory protein AfsR

MacromoleculeName: Regulatory protein AfsR / type: protein_or_peptide / ID: 8 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 31.479572 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MDGGPRVPEQ RRPGFPAEEE ESGALRFGVL GPVRAWRDGE TLATGSPQQR ALLAALLLRE GRTATAGEL IDALWGEEPP SQALAAVRTY ASRLRKVLDP GVLVSESGGY AVRGLAEGAL DLARAQDLAS AAEKARSAGD L CHARDLLR ...String:
MGSSHHHHHH SSGLVPRGSH MDGGPRVPEQ RRPGFPAEEE ESGALRFGVL GPVRAWRDGE TLATGSPQQR ALLAALLLRE GRTATAGEL IDALWGEEPP SQALAAVRTY ASRLRKVLDP GVLVSESGGY AVRGLAEGAL DLARAQDLAS AAEKARSAGD L CHARDLLR RALDLWDGEV LAGVPGPYAQ TQRVRLGEWR LQLLETRLDM DLDQGCHAEA VSELTALTAA HPLRERLREL LM LALYRSG RQAEALAVYA DTRRLLADEL GVDPRPGLQE LQQRILQADP ALA

UniProtKB: Regulatory protein AfsR

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Macromolecule #9: DNA (65-MER)

MacromoleculeName: DNA (65-MER) / type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 19.863656 KDa
SequenceString: (DG)(DT)(DA)(DG)(DC)(DC)(DG)(DG)(DA)(DG) (DC)(DG)(DT)(DT)(DC)(DA)(DG)(DC)(DG)(DT) (DT)(DC)(DG)(DT)(DT)(DT)(DA)(DT)(DC) (DT)(DC)(DC)(DC)(DC)(DC)(DT)(DG)(DG)(DC) (DA) (DC)(DT)(DG)(DT)(DC)(DA) ...String:
(DG)(DT)(DA)(DG)(DC)(DC)(DG)(DG)(DA)(DG) (DC)(DG)(DT)(DT)(DC)(DA)(DG)(DC)(DG)(DT) (DT)(DC)(DG)(DT)(DT)(DT)(DA)(DT)(DC) (DT)(DC)(DC)(DC)(DC)(DC)(DT)(DG)(DG)(DC) (DA) (DC)(DT)(DG)(DT)(DC)(DA)(DT)(DC) (DT)(DC)(DC)(DG)(DT)(DC)(DA)(DG)(DA)(DC) (DC)(DG) (DT)(DC)(DG)(DC)(DA)

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Macromolecule #10: DNA (65-MER)

MacromoleculeName: DNA (65-MER) / type: dna / ID: 10 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 20.086846 KDa
SequenceString: (DT)(DG)(DC)(DG)(DA)(DC)(DG)(DG)(DT)(DC) (DT)(DG)(DA)(DC)(DG)(DC)(DT)(DC)(DT)(DA) (DC)(DA)(DC)(DA)(DG)(DT)(DG)(DC)(DC) (DA)(DG)(DG)(DG)(DG)(DG)(DA)(DG)(DA)(DT) (DA) (DA)(DA)(DC)(DG)(DA)(DA) ...String:
(DT)(DG)(DC)(DG)(DA)(DC)(DG)(DG)(DT)(DC) (DT)(DG)(DA)(DC)(DG)(DC)(DT)(DC)(DT)(DA) (DC)(DA)(DC)(DA)(DG)(DT)(DG)(DC)(DC) (DA)(DG)(DG)(DG)(DG)(DG)(DA)(DG)(DA)(DT) (DA) (DA)(DA)(DC)(DG)(DA)(DA)(DC)(DG) (DC)(DT)(DG)(DA)(DA)(DC)(DG)(DC)(DT)(DC) (DC)(DG) (DG)(DC)(DT)(DA)(DC)

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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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Macromolecule #12: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 12 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTRISTRIS
50.0 mMKClKCl
3.0 mMDTTDTT
5.0 mMMgCl2MgCl2
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 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: 193644
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 95223
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8hvr:
Cryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter

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