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- EMDB-50511: Cryo-EM structure of Mycobacterium tuberculosis sigma-B RNA polym... -

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Basic information

Entry
Database: EMDB / ID: EMD-50511
TitleCryo-EM structure of Mycobacterium tuberculosis sigma-B RNA polymerase bound to -10 promoter element ssDNA oligo - sigma-B udocked conformation
Map dataprimary map
Sample
  • Complex: RNA polymerase holoenzyme bound to -10 promoter element ssDNA oligo
    • 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 SigB
    • DNA: DNA (17-MER)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsRNA polymerase / sigma factor / SigB / stress response / -10 promoter element / promoter recognition / tuberculosis / TRANSCRIPTION
Function / homology
Function and homology information


RNA polymerase core enzyme binding / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / DNA-directed RNA polymerase activity / ribonucleoside binding ...RNA polymerase core enzyme binding / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / DNA-directed RNA polymerase activity / ribonucleoside binding / DNA-directed RNA polymerase / response to heat / response to hypoxia / protein dimerization activity / response to xenobiotic stimulus / response to antibiotic / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Sigma-70 factors family signature 1. / : / 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 ...Sigma-70 factors family signature 1. / : / 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 / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / 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 Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, RBP11-like subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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 SigB / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.48 Å
AuthorsBrodolin K / Blaise M
Funding support France, 3 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-20-CE44-0020-01 France
Agence Nationale de la Recherche (ANR)ANR-16-CE11-0025 France
Instruct-ERIC Center (Strasbourg Centre)19348 France
CitationJournal: Nucleic Acids Res / Year: 2018
Title: RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase.
Authors: Ayyappasamy Sudalaiyadum Perumal / Rishi Kishore Vishwakarma / Yangbo Hu / Zakia Morichaud / Konstantin Brodolin /
Abstract: The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex ...The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex (RPo). Here, we explored the influence of promoter motifs on RbpA-mediated activation of MtbRNAP containing the stress-response σB subunit. We show that both the 'extended -10' promoter motif (T-17G-16T-15G-14) and RbpA stabilized RPo and allowed promoter opening at suboptimal temperatures. Furthermore, in the presence of the T-17G-16T-15G-14 motif, RbpA was dispensable for RNA synthesis initiation, while exerting a stabilization effect on RPo. On the other hand, RbpA compensated for the lack of sequence-specific interactions of domains 3 and 4 of σB with the extended -10 and the -35 motifs, respectively. Mutations of the positively charged residues K73, K74 and R79 in RbpA basic linker (BL) had little effect on RPo formation, but affected MtbRNAP capacity for de novo transcription initiation. We propose that RbpA stimulates transcription by strengthening the non-specific interaction of the σ subunit with promoter DNA upstream of the -10 element, and by indirectly optimizing MtbRNAP interaction with initiation substrates. Consequently, RbpA renders MtbRNAP promiscuous in promoter selection, thus compensating for the weak conservation of the -35 motif in mycobacteria.
History
DepositionMay 31, 2024-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateMay 7, 2025-
Current statusMay 7, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50511.png

Downloads & links

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Map

FileDownload / File: emd_50511.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprimary map
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 360 pix.
= 310.32 Å		0.86 Å/pix.
x 360 pix.
= 310.32 Å		0.86 Å/pix.
x 360 pix.
= 310.32 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.862 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.115
Minimum - Maximum-0.5259665 - 0.978114
Average (Standard dev.)0.002375959 (±0.025733074)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 310.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50511_msk_1.map
Projections & Slices
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Additional map: Raw map

Fileemd_50511_additional_1.map
AnnotationRaw map
Projections & Slices
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Histogram (log scale)

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Half map: half-map A

Fileemd_50511_half_map_1.map
Annotationhalf-map A
Projections & Slices
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Histogram (log scale)

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Half map: half-map B

Fileemd_50511_half_map_2.map
Annotationhalf-map B
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Sample components

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Entire : RNA polymerase holoenzyme bound to -10 promoter element ssDNA oligo

EntireName: RNA polymerase holoenzyme bound to -10 promoter element ssDNA oligo
Components
  • Complex: RNA polymerase holoenzyme bound to -10 promoter element ssDNA oligo
    • 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 SigB
    • DNA: DNA (17-MER)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: RNA polymerase holoenzyme bound to -10 promoter element ssDNA oligo

SupramoleculeName: RNA polymerase holoenzyme bound to -10 promoter element ssDNA oligo
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 406 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: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 37.745328 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLISQRPTLS EDVLTDNRSQ FVIEPLEPGF GYTLGNSLRR TLLSSIPGAA VTSIRIDGVL HEFTTVPGVK EDVTEIILNL KSLVVSSEE DEPVTMYLRK QGPGEVTAGD IVPPAGVTVH NPGMHIATLN DKGKLEVELV VERGRGYVPA VQNRASGAEI G RIPVDSIY ...String:
MLISQRPTLS EDVLTDNRSQ FVIEPLEPGF GYTLGNSLRR TLLSSIPGAA VTSIRIDGVL HEFTTVPGVK EDVTEIILNL KSLVVSSEE DEPVTMYLRK QGPGEVTAGD IVPPAGVTVH NPGMHIATLN DKGKLEVELV VERGRGYVPA VQNRASGAEI G RIPVDSIY SPVLKVTYKV DATRVEQRTD FDKLILDVET KNSISPRDAL ASAGKTLVEL FGLARELNVE AEGIEIGPSP AE ADHIASF ALPIDDLDLT VRSYNCLKRE GVHTVGELVA RTESDLLDIR NFGQKSIDEV KIKLHQLGLS LKDSPPSFDP SEV AGYDVA TGTWSTEGAY DEQDYAETEQ L

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: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 129.602344 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MVLADSRQSK TAASPSPSRP QSSSNNSVPG APNRVSFAKL REPLEVPGLL DVQTDSFEWL IGSPRWRESA AERGDVNPVG GLEEVLYEL SPIEDFSGSM SLSFSDPRFD DVKAPVDECK DKDMTYAAPL FVTAEFINNN TGEIKSQTVF MGDFPMMTEK G TFIINGTE ...String:
MVLADSRQSK TAASPSPSRP QSSSNNSVPG APNRVSFAKL REPLEVPGLL DVQTDSFEWL IGSPRWRESA AERGDVNPVG GLEEVLYEL SPIEDFSGSM SLSFSDPRFD DVKAPVDECK DKDMTYAAPL FVTAEFINNN TGEIKSQTVF MGDFPMMTEK G TFIINGTE RVVVSQLVRS PGVYFDETID KSTDKTLHSV KVIPSRGAWL EFDVDKRDTV GVRIDRKRRQ PVTVLLKALG WT SEQIVER FGFSEIMRST LEKDNTVGTD EALLDIYRKL RPGEPPTKES AQTLLENLFF KEKRYDLARV GRYKVNKKLG LHV GEPITS STLTEEDVVA TIEYLVRLHE GQTTMTVPGG VEVPVETDDI DHFGNRRLRT VGELIQNQIR VGMSRMERVV RERM TTQDV EAITPQTLIN IRPVVAAIKE FFGTSQLSQF MDQNNPLSGL THKRRLSALG PGGLSRERAG LEVRDVHPSH YGRMC PIET PEGPNIGLIG SLSVYARVNP FGFIETPYRK VVDGVVSDEI VYLTADEEDR HVVAQANSPI DADGRFVEPR VLVRRK AGE VEYVPSSEVD YMDVSPRQMV SVATAMIPFL EHDDANRALM GANMQRQAVP LVRSEAPLVG TGMELRAAID AGDVVVA EE SGVIEEVSAD YITVMHDNGT RRTYRMRKFA RSNHGTCANQ CPIVDAGDRV EAGQVIADGP CTDDGEMALG KNLLVAIM P WEGHNYEDAI ILSNRLVEED VLTSIHIEEH EIDARDTKLG AEEITRDIPN ISDEVLADLD ERGIVRIGAE VRDGDILVG KVTPKGETEL TPEERLLRAI FGEKAREVRD TSLKVPHGES GKVIGIRVFS REDEDELPAG VNELVRVYVA QKRKISDGDK LAGRHGNKG VIGKILPVED MPFLADGTPV DIILNTHGVP RRMNIGQILE THLGWCAHSG WKVDAAKGVP DWAARLPDEL L EAQPNAIV STPVFDGAQE AELQGLLSCT LPNRDGDVLV DADGKAMLFD GRSGEPFPYP VTVGYMYIMK LHHLVDDKIH AR STGPYSM ITQQPLGGKA QFGGQRFGEM ECWAMQAYGA AYTLQELLTI KSDDTVGRVK VYEAIVKGEN IPEPGIPESF KVL LKELQS LCLNVEVLSS DGAAIELREG EDEDLERAAA NLGINLSRNE SASVEDLA

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: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 147.438344 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: VNFFDELRIG LATAEDIRQW SYGEVKKPET INYRTLKPEK DGLFCEKIFG PTRDWECYCG KYKRVRFKGI ICERCGVEVT RAKVRRERM GHIELAAPVT HIWYFKGVPS RLGYLLDLAP KDLEKIIYFA AYVITSVDEE MRHNELSTLE AEMAVERKAV E DQRDGELE ...String:
VNFFDELRIG LATAEDIRQW SYGEVKKPET INYRTLKPEK DGLFCEKIFG PTRDWECYCG KYKRVRFKGI ICERCGVEVT RAKVRRERM GHIELAAPVT HIWYFKGVPS RLGYLLDLAP KDLEKIIYFA AYVITSVDEE MRHNELSTLE AEMAVERKAV E DQRDGELE ARAQKLEADL AELEAEGAKA DARRKVRDGG EREMRQIRDR AQRELDRLED IWSTFTKLAP KQLIVDENLY RE LVDRYGE YFTGAMGAES IQKLIENFDI DAEAESLRDV IRNGKGQKKL RALKRLKVVA AFQQSGNSPM GMVLDAVPVI PPE LRPMVQ LDGGRFATSD LNDLYRRVIN RNNRLKRLID LGAPEIIVNN EKRMLQESVD ALFDNGRRGR PVTGPGNRPL KSLS DLLKG KQGRFRQNLL GKRVDYSGRS VIVVGPQLKL HQCGLPKLMA LELFKPFVMK RLVDLNHAQN IKSAKRMVER QRPQV WDVL EEVIAEHPVL LNRAPTLHRL GIQAFEPMLV EGKAIQLHPL VCEAFNADFD GDQMAVHLPL SAEAQAEARI LMLSSN NIL SPASGRPLAM PRLDMVTGLY YLTTEVPGDT GEYQPASGDH PETGVYSSPA EAIMAADRGV LSVRAKIKVR LTQLRPP VE IEAELFGHSG WQPGDAWMAE TTLGRVMFNE LLPLGYPFVN KQMHKKVQAA IINDLAERYP MIVVAQTVDK LKDAGFYW A TRSGVTVSMA DVLVPPRKKE ILDHYEERAD KVEKQFQRGA LNHDERNEAL VEIWKEATDE VGQALREHYP DDNPIITIV DSGATGNFTQ TRTLAGMKGL VTNPKGEFIP RPVKSSFREG LTVLEYFINT HGARKGLADT ALRTADSGYL TRRLVDVSQD VIVREHDCQ TERGIVVELA ERAPDGTLIR DPYIETSAYA RTLGTDAVDE AGNVIVERGQ DLGDPEIDAL LAAGITQVKV R SVLTCATS TGVCATCYGR SMATGKLVDI GEAVGIVAAQ SIGEPGTQLT MRTFHQGGVG EDITGGLPRV QELFEARVPR GK APIADVT GRVRLEDGER FYKITIVPDD GGEEVVYDKI SKRQRLRVFK HEDGSERVLS DGDHVEVGQQ LMEGSADPHE VLR VQGPRE VQIHLVREVQ EVYRAQGVSI HDKHIEVIVR QMLRRVTIID SGSTEFLPGS LIDRAEFEAE NRRVVAEGGE PAAG RPVLM GITKASLATD SWLSAASFQE TTRVLTDAAI NCRSDKLNGL KENVIIGKLI PAGTGINRYR NIAVQPTEEA RAAAY TIPS YEDQYYSPDF GAATGAAVPL DDYGYSDYRH HHHHH

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: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 11.85114 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSISQSDASL AAVPAVDQFD PSSGASGGYD TPLGITNPPI DELLDRVSSK YALVIYAAKR ARQINDYYNQ LGEGILEYVG PLVEPGLQE KPLSIALREI HADLLEHTEG E

UniProtKB: DNA-directed RNA polymerase subunit omega

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Macromolecule #5: RNA polymerase sigma factor SigB

MacromoleculeName: RNA polymerase sigma factor SigB / type: protein_or_peptide / ID: 5
Details: Addition of twenty N-terminal residues (MGSSHHHHHHSSGLVPRGSH) including 6xHIS tag
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 38.572773 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MADAPTRATT SRVDSDLDAQ SPAADLVRVY LNGIGKTALL NAAGEVELAK RIEAGLYAEH LLETRKRLG ENRKRDLAAV VRDGEAARRH LLEANLRLVV SLAKRYTGRG MPLLDLIQEG NLGLIRAMEK FDYTKGFKFS T YATWWIRQ ...String:
MGSSHHHHHH SSGLVPRGSH MADAPTRATT SRVDSDLDAQ SPAADLVRVY LNGIGKTALL NAAGEVELAK RIEAGLYAEH LLETRKRLG ENRKRDLAAV VRDGEAARRH LLEANLRLVV SLAKRYTGRG MPLLDLIQEG NLGLIRAMEK FDYTKGFKFS T YATWWIRQ AITRGMADQS RTIRLPVHLV EQVNKLARIK REMHQHLGRE ATDEELAAES GIPIDKINDL LEHSRDPVSL DM PVGSEEE APLGDFIEDA EAMSAENAVI AELLHTDIRS VLATLDEREH QVIRLRFGLD DGQPRTLDQI GKLFGLSRER VRQ IERDVM SKLRHGERAD RLRSYAS

UniProtKB: RNA polymerase sigma factor SigB

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Macromolecule #6: DNA (17-MER)

MacromoleculeName: DNA (17-MER) / type: dna / ID: 6 / Details: synthetic oligonucleotide / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 5.297444 KDa
SequenceString:
(DT)(DG)(DC)(DG)(DT)(DA)(DT)(DA)(DA)(DT) (DG)(DT)(DG)(DT)(DG)(DG)(DA)

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / 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

Concentration6 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMKClpotassium chloride
5.0 mMMgCl2magnesium chloride
2.0 mMC4H10O2S2dithiothreitol

Details: 8 mM CHAPSO added before vitrificaion
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 291.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 9202 / Average exposure time: 1.997 sec. / Average electron dose: 55.735 e/Å2 / Details: images were collected in super-resolution mode
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: 0.01 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
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: 695496
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 67957
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7pp4

source_name: PDB, initial_model_type: experimental model
chain_id: f, residue_range: 159-323, source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9fjs:
Cryo-EM structure of Mycobacterium tuberculosis sigma-B RNA polymerase bound to -10 promoter element ssDNA oligo - sigma-B undocked conformation

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