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Yorodumi- PDB-7qxi: Cryo-EM structure of RNA polymerase-sigma54 holo enzyme with prom... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7qxi | |||||||||
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Title | Cryo-EM structure of RNA polymerase-sigma54 holo enzyme with promoter DNA closed complex | |||||||||
Components |
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Keywords | TRANSCRIPTION / Cryo EM / RNA polymerase / transcription close complex / sigma54 | |||||||||
Function / homology | Function and homology information RNA polymerase complex / DNA-binding transcription activator activity / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility / nitrate assimilation ...RNA polymerase complex / DNA-binding transcription activator activity / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility / nitrate assimilation / nucleotidyltransferase activity / transcription elongation factor complex / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / transcription antitermination / cell motility / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Escherichia coli K-12 (bacteria) Klebsiella pneumoniae (bacteria) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | |||||||||
Authors | Ye, F.Z. / Zhang, X.D. | |||||||||
Funding support | United Kingdom, 2items
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Citation | Journal: Sci Adv / Year: 2022 Title: Mechanisms of DNA opening revealed in AAA+ transcription complex structures. Authors: Fuzhou Ye / Forson Gao / Xiaojiao Liu / Martin Buck / Xiaodong Zhang / Abstract: Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter ...Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter complex, where DNA is opened up. In bacteria, RNAP relies on σ factors for its promoter specificities. Using a special form of sigma factor (σ), which forms a stable closed complex and requires its activator that belongs to the AAA+ ATPases (ATPases associated with diverse cellular activities), we obtained cryo-electron microscopy structures of transcription initiation complexes that reveal a previously unidentified process of DNA melting opening. The σ amino terminus threads through the locally opened up DNA and then becomes enclosed by the AAA+ hexameric ring in the activator-bound intermediate complex. Our structures suggest how ATP hydrolysis by the AAA+ activator could remove the σ inhibition while helping to open up DNA, using σ amino-terminal peptide as a pry bar. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7qxi.cif.gz | 677.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7qxi.ent.gz | 537.3 KB | Display | PDB format |
PDBx/mmJSON format | 7qxi.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/qx/7qxi ftp://data.pdbj.org/pub/pdb/validation_reports/qx/7qxi | HTTPS FTP |
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-Related structure data
Related structure data | 14200MC 7qv9C 7qwpC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE
#1: Protein | Mass: 36558.680 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Plasmid: pGEXABC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Escherichia coli / References: UniProt: P0A7Z4, DNA-directed RNA polymerase #2: Protein | | Mass: 150820.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950 Plasmid: pGEXABC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Escherichia coli / References: UniProt: P0A8V2, DNA-directed RNA polymerase #3: Protein | | Mass: 155366.781 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: rpoC, tabB, b3988, JW3951 / Plasmid: pGEXABC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Escherichia coli / References: UniProt: P0A8T7, DNA-directed RNA polymerase #4: Protein | | Mass: 10249.547 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: rpoZ, b3649, JW3624 / Plasmid: paCYCDuet-omega / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Escherichia coli / References: UniProt: P0A800, DNA-directed RNA polymerase |
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-Protein , 1 types, 1 molecules M
#5: Protein | Mass: 56165.027 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Klebsiella pneumoniae (bacteria) Gene: rpoN, B4U61_10775, B5L96_24890, BANRA_01956, BANRA_03272, BANRA_04880, BL124_00020515, BN49_0508, BS595_02095, BVX91_19500, C3F39_17700, DBX64_03930, DD583_08830, DDJ63_09965, DRB11_15360, ...Gene: rpoN, B4U61_10775, B5L96_24890, BANRA_01956, BANRA_03272, BANRA_04880, BL124_00020515, BN49_0508, BS595_02095, BVX91_19500, C3F39_17700, DBX64_03930, DD583_08830, DDJ63_09965, DRB11_15360, EHZ17_05145, FXN67_08250, G5637_17550, G7Z27_02650, GJJ08_002620, GNG14_08115, GTH21_20225, NCTC13465_03419, NCTC3279_03959, NCTC5047_03164, SAMEA3499874_03190, SAMEA3499901_03507, SAMEA3512100_03793, SAMEA3538828_04129, SAMEA3649466_03329, SAMEA3649648_01442, SAMEA3649733_03935, SAMEA3649758_00080, SAMEA3720909_03353, SAMEA3727643_03388, SAMEA3727679_00080, SAMEA3729663_03804, SAMEA4364603_00630 Plasmid: pET28b-sigma54 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0N9UTC1 |
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-DNA chain , 2 types, 2 molecules NT
#6: DNA chain | Mass: 19439.387 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (bacteria) |
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#7: DNA chain | Mass: 19404.410 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (bacteria) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component |
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Molecular weight | Value: 0.5 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 8 Details: 20 mM Tris-HCl pH 8.0, 150 mM KCl, 10 mM MgCl2,8 mM CHAPSO | ||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: The sample was mxied in vitro in an order to form complex | ||||||||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2 | ||||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1400 nm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 4.1 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14780 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 2200000 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29321 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: RECIPROCAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5NSR |