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- EMDB-35130: Clostridium thermocellum RNA polymerase transcription open comple... -

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Entry
Database: EMDB / ID: EMD-35130
TitleClostridium thermocellum RNA polymerase transcription open complex with SigI1 and its promoter
Map data
Sample
  • Complex: Clostridium thermocellum RNA polymerase transcription open complex with SigI1 its 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 SigI1
    • DNA: DNA (80-mer)
    • DNA: DNA (80-mer)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsTRANSCRIPTION OPEN COMPLEX / SIGI / TRANSCRIPTION / TRANSCRIPTION-DNA complex
Biological speciesAcetivibrio thermocellus DSM 1313 (bacteria) / Acetivibrio thermocellus DSM1313 (bacteria) / Acetivibrio thermocellus (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 3.03 Å
AuthorsLi J / Zhang H / Li D / Feng Y / Zhu P
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32070125 China
CitationJournal: Nat Commun / Year: 2023
Title: Structure of the transcription open complex of distinct σ factors.
Authors: Jie Li / Haonan Zhang / Dongyu Li / Ya-Jun Liu / Edward A Bayer / Qiu Cui / Yingang Feng / Ping Zhu /
Abstract: Bacterial σ factors of the σ-family are widespread in Bacilli and Clostridia and are involved in the heat shock response, iron metabolism, virulence, and carbohydrate sensing. A multiplicity of σ ...Bacterial σ factors of the σ-family are widespread in Bacilli and Clostridia and are involved in the heat shock response, iron metabolism, virulence, and carbohydrate sensing. A multiplicity of σ paralogues in some cellulolytic bacteria have been shown to be responsible for the regulation of the cellulosome, a multienzyme complex that mediates efficient cellulose degradation. Here, we report two structures at 3.0 Å and 3.3 Å of two transcription open complexes formed by two σ factors, SigI1 and SigI6, respectively, from the thermophilic, cellulolytic bacterium, Clostridium thermocellum. These structures reveal a unique, hitherto-unknown recognition mode of bacterial transcriptional promoters, both with respect to domain organization and binding to promoter DNA. The key characteristics that determine the specificities of the σ paralogues were further revealed by comparison of the two structures. Consequently, the σ factors represent a distinct set of the σ-family σ factors, thus highlighting the diversity of bacterial transcription.
History
DepositionJan 13, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35130.png

Downloads & links

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Map

FileDownload / File: emd_35130.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.0442
Minimum - Maximum-0.019557968 - 1.9130006
Average (Standard dev.)0.0022479068 (±0.03324933)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 291.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35130_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35130_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35130_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Clostridium thermocellum RNA polymerase transcription open comple...

EntireName: Clostridium thermocellum RNA polymerase transcription open complex with SigI1 its promoter
Components
  • Complex: Clostridium thermocellum RNA polymerase transcription open complex with SigI1 its 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 SigI1
    • DNA: DNA (80-mer)
    • DNA: DNA (80-mer)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Clostridium thermocellum RNA polymerase transcription open comple...

SupramoleculeName: Clostridium thermocellum RNA polymerase transcription open complex with SigI1 its promoter
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Acetivibrio thermocellus DSM 1313 (bacteria)

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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: Acetivibrio thermocellus DSM 1313 (bacteria) / Strain: DSM1313
Molecular weightTheoretical: 35.071125 KDa
SequenceString: MIEIEKPKIE CVVCSEDNRY GKFVVEPLER GYGITLGNSL RRILLSSLPG VAVTSIKIDG VLHEFSTIPG VIEDVTEIIL NIKELSLNF HGEGPKVIYI DAEGEGEVKA KDIKADADVE ILNPEHKIAT LSGDHRLYME MTIDKGRGYV SAEKNKHPGQ P IGVIPVDS ...String:
MIEIEKPKIE CVVCSEDNRY GKFVVEPLER GYGITLGNSL RRILLSSLPG VAVTSIKIDG VLHEFSTIPG VIEDVTEIIL NIKELSLNF HGEGPKVIYI DAEGEGEVKA KDIKADADVE ILNPEHKIAT LSGDHRLYME MTIDKGRGYV SAEKNKHPGQ P IGVIPVDS IFTPVHKVNY TVENTRVGQV TDYDKLTLEV WTNGSIKPDE AISLGAKILS EHLNLFIDLS DNAKNAEIMV EK EETKKEK VLEMTIEELD LSVRSYNCLK RAGINTVEDL ISRTEEDMMK VRNLGRKSLE EVVNKLKALG LSLAPSED

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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: Acetivibrio thermocellus DSM1313 (bacteria) / Strain: DSM1313
Molecular weightTheoretical: 140.110359 KDa
SequenceString: MVHPVKLGRN VRMSYSKIDE VIDMPNLIEI QKNSYEQFLK EGFKEVFKDV NPITDYTGNL ILEFVDYSLD EPPKYSVDEC KERDATYAA PLKVKVRLIN KETGEVKEQE IFMGDFPLMT ETGTFIINGA ERVIVSQLVR SPGIYYAMKI DKAGKQLFSN T VIPNRGAW ...String:
MVHPVKLGRN VRMSYSKIDE VIDMPNLIEI QKNSYEQFLK EGFKEVFKDV NPITDYTGNL ILEFVDYSLD EPPKYSVDEC KERDATYAA PLKVKVRLIN KETGEVKEQE IFMGDFPLMT ETGTFIINGA ERVIVSQLVR SPGIYYAMKI DKAGKQLFSN T VIPNRGAW LEYETDSNDV LSVRIDRTRK LPLTVLVRAL GYGTDLEITE LFGEDERILA TIQKDSTKTE EEGLLEIYKR LR PGEPPTV ESAKALLHGL FFDPKRYDLA KPGRFKFNKK LSIAARIHGF IAGENIKDPD TGEIIVAEGE TISREKAETI QNA GVNTVI LRVDGKNVKV IGNDMVDIKR YVDFDPKEIG INEKVKRDVL MEILEEYKGK GDDAIKKALQ ERIDDLIPKH ITKE DIISS ISYIIGLSYG IGSTDDIDHL GNRRLRSVGE LLQNQFRIGL SRMERVVRER MTIQDLDVVT PQALINIRPV AAAIK EFFG SSQLSQFMDQ TNPLAELTHK RRLSALGPGG LSRERAGFEV RDVHHSHYGR MCPIETPEGP NIGLIGSLST YARVNE YGF IETPYRKVSK EEPGKVTNEI VYLTADEEDE YIIAQANEPL DEEGRFISNK VVCRYKEEFI EVDPSKIDFM DVSPKQI VS VATSMIPFLE NDDANRALMG ANMQRQAVPL IKTESPIVGT GIEYRAARDS GVVILAKNPG VVEKVTANEI IIRTKDGK R DTYKLLKYMR SNQGTCINQR PIVKKGEEVE AGDVIADGPS TDNGEIALGK NVLVGFMTWE GYNYEDAILI SERLVKDDV FTSIHIEEYE AEARDTKLGP EDITREIPNV SEDALKDLNS EGIIRIGAEV RAGDILVGKV TPKGETELTA EERLLRAIFG EKAREVRDT SLRVPHGESG IVVDVKIFTR ENGDELAPGV NKLVRVYVAQ KRKISVGDKM AGRHGNKGVI SRILPVEDMP F LPDGTPLD IVLNPLGVPS RMNIGQVLEV HLGYAAKALG WKVATPVFDG ATEEDIVQTL RKAGLAEDGK SILYDGRTGE PF ENRVTVG YMYMLKLAHL VDDKIHARST GPYSLVTQQP LGGKAQFGGQ RFGEMEVWAL EAYGAAYTLQ EILTVKSDDV VGR VKTYEA IVKGENVPEP GIPESFKVLI KELQSLCLDV KVYSEEQEEI AIKESVEDDL EELNVNIEGR EDEVNFNEFN DIGE EITDE DLEVEDFDLQ DLNDDDINPD DTIDAELDDN LFDDDFDDTF DDDDL

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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: Acetivibrio thermocellus DSM 1313 (bacteria) / Strain: DSM1313
Molecular weightTheoretical: 132.698844 KDa
SequenceString: MGSSHHHHHH HHHHSGSGSG SGSGFELNNF DSIRIGLASP EKIREWSRGE VKKPETINYR TLKPERDGLF CERIFGPQKD WECHCGKYK RIRYKGIVCD RCGVEVTRSK VRRERMGHIE LAAPVSHIWY FKGIPSRMGL LLDMSPRALE KILYFAAYVV I DPGQTPLS ...String:
MGSSHHHHHH HHHHSGSGSG SGSGFELNNF DSIRIGLASP EKIREWSRGE VKKPETINYR TLKPERDGLF CERIFGPQKD WECHCGKYK RIRYKGIVCD RCGVEVTRSK VRRERMGHIE LAAPVSHIWY FKGIPSRMGL LLDMSPRALE KILYFAAYVV I DPGQTPLS KKQILSEKEY RDSLEKFGPK FRAGMGAEAV RELLQEINLD ELSAELREEI KQSTGQKRVR AIKRLEVVEA FR QSQNKPE WMILDVIPVI PPELRPMVQL DGGRFATSDL NDLYRRVINR NNRLKRLLDL GAPDIIVRNE KRMLQEAVDA LID NGRRGR PVTGPGNRPL KSLSDMLKGK QGRFRQNLLG KRVDYSGRSV IVVGPELKIY QCGLPKEMAL ELFKPFVMKK LVND GLAHN IKSAKRMVER VRNEVWDVLE EVIKEHPVLL NRAPTLHRLG IQAFEPVLVE GRALKLHPLV CTAYNADFDG DQMAI HVPL SAEAQAEARF LMLSANNLLK PQDGKPVAVP TQDMVLGSYY LTILKEGAKG EGRVFTSMDE AVMAYDNGEI ELHSKI KVR MKRVVDGVEK SKIIETTLGR LIFNEAIPQD LGFVDRSDPD KIFDLEVDFL VGKNELKKII DKSIKVHGTT KTAILLD KI KELGFKYSTK GAITISISDM VIPEVKAKYI KETEEKIEKI TKQYKRGLIS DEERYNSVIA AWTEASENIT RALINNLD R FNPVYMMSQS GARGNINQIK QLAGMRGLMA DTSGKTIEFP IKANFREGLT VMEFFISTHG ARKGLADTAL RTADSGYLT RRLVDVSQDV IVRETDCGTR KGIEVTDIKD GNEVIEELSE RIIGRYPVGN IVHPETGEII VEAGRMITDQ DAEKIVKAGI KKVRIRSVL TCHSEYGVCA KCYGANLATG EECNVGEAVG IIAAQSIGEP GTQLTMRTFH TGGVAGEDIT QGLPRVEELF E ARKPKGLA IISEIKGTVK ISETKKKREI VVTSEDGETR SYLIPYGSRI KVSDGDQVEA GDELTEGSVN PHDILKIKGV EA VQTYLVH EVQKVYRMQG VDINDKHIEV IVRQMLRKVK VEDPGDTSLL PGGLVDVFDF EEENAKAIAE GKKPAVAKRA LLG ITKAAL ATDSFLSAAS FQETTRVLTE AAIKGKVDPL VGLKENVIIG KLIPAGTGMS RYKDITISTV TE

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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: Acetivibrio thermocellus DSM1313 (bacteria) / Strain: DSM1313
Molecular weightTheoretical: 8.783143 KDa
SequenceString:
MKEKKERVSS MIEPSINSLL EKVDSRYTLV VATAKRARQL TDGANKLTNC ESDKPVTVAI NEINENKITY IRTKSGIK

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Macromolecule #5: RNA polymerase sigma factor SigI1

MacromoleculeName: RNA polymerase sigma factor SigI1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Acetivibrio thermocellus DSM1313 (bacteria) / Strain: DSM1313
Molecular weightTheoretical: 30.714949 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SMEVRKINTH NREKKLDDIV YDDFISTLRQ IKEGNHQLRE EFISEYKPFI LKVTSNATGK YIDTRNSDEF SIALSAFNEA IDKFDIEKG YNFFLFSEQV IRRRLIDYSR SNKDDKEYPF SFFDDEYFYN NEKLLSKSYI GFEDIEARED IEELKKKLQE F GITFLDLV ...String:
SMEVRKINTH NREKKLDDIV YDDFISTLRQ IKEGNHQLRE EFISEYKPFI LKVTSNATGK YIDTRNSDEF SIALSAFNEA IDKFDIEKG YNFFLFSEQV IRRRLIDYSR SNKDDKEYPF SFFDDEYFYN NEKLLSKSYI GFEDIEARED IEELKKKLQE F GITFLDLV LNVPKHRDSR QLCIRLAKML AEDEQMYNAL MKNKNIPRNE LKKKAKVHGR TIGNNRKYII ALCLIFRSNL NL SKRYLEY YTMDGESDLI

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Macromolecule #6: DNA (80-mer)

MacromoleculeName: DNA (80-mer) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Acetivibrio thermocellus (bacteria)
Molecular weightTheoretical: 24.897059 KDa
SequenceString: (DA)(DG)(DA)(DG)(DC)(DC)(DG)(DA)(DT)(DA) (DT)(DT)(DA)(DA)(DT)(DC)(DG)(DA)(DT)(DA) (DA)(DT)(DA)(DT)(DA)(DC)(DA)(DC)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DG)(DC)(DA)(DG) (DA) (DT)(DG)(DT)(DA)(DT)(DA) ...String:
(DA)(DG)(DA)(DG)(DC)(DC)(DG)(DA)(DT)(DA) (DT)(DT)(DA)(DA)(DT)(DC)(DG)(DA)(DT)(DA) (DA)(DT)(DA)(DT)(DA)(DC)(DA)(DC)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DG)(DC)(DA)(DG) (DA) (DT)(DG)(DT)(DA)(DT)(DA)(DC)(DG) (DA)(DA)(DG)(DT)(DA)(DA)(DT)(DC)(DT)(DA) (DC)(DT) (DG)(DA)(DA)(DG)(DG)(DG)(DA) (DG)(DA)(DA)(DT)(DG)(DA)(DT)(DC)(DT)(DG) (DG)(DT)(DG)

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Macromolecule #7: DNA (80-mer)

MacromoleculeName: DNA (80-mer) / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Acetivibrio thermocellus (bacteria)
Molecular weightTheoretical: 24.401629 KDa
SequenceString: (DC)(DA)(DC)(DC)(DA)(DG)(DA)(DT)(DC)(DA) (DT)(DT)(DC)(DT)(DC)(DC)(DC)(DT)(DA)(DG) (DT)(DC)(DA)(DT)(DC)(DT)(DA)(DA)(DA) (DC)(DT)(DT)(DC)(DG)(DT)(DA)(DT)(DA)(DC) (DA) (DT)(DC)(DT)(DG)(DC)(DT) ...String:
(DC)(DA)(DC)(DC)(DA)(DG)(DA)(DT)(DC)(DA) (DT)(DT)(DC)(DT)(DC)(DC)(DC)(DT)(DA)(DG) (DT)(DC)(DA)(DT)(DC)(DT)(DA)(DA)(DA) (DC)(DT)(DT)(DC)(DG)(DT)(DA)(DT)(DA)(DC) (DA) (DT)(DC)(DT)(DG)(DC)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DG)(DT)(DG)(DT)(DA)(DT) (DA)(DT) (DT)(DA)(DT)(DC)(DG)(DA)(DT) (DT)(DA)(DA)(DT)(DA)(DT)(DC)(DG)(DG)(DC) (DT)(DC)(DT)

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration23.54 mg/mL
BufferpH: 8
Details: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM DTT, 0.2 mM EDTA, 5% (v/v) glycerol, 10 mM MgCl2
StainingType: NEGATIVE / Material: Uranyl Acetate
VitrificationCryogen name: ETHANE
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-32 / Average exposure time: 0.25 sec. / Average electron dose: 1.875 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 22500
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

Startup modelType of model: OTHER
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 150741
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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