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- PDB-8gzh: Cryo-EM structure of Synechocystis sp. PCC 6803 CTP-bound RPitc -

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Basic information

Entry
Database: PDB / ID: 8gzh
TitleCryo-EM structure of Synechocystis sp. PCC 6803 CTP-bound RPitc
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 5
  • Nontemplate strand DNA
  • RNA (5'-R(*CP*CP*GP*A)-3')
  • RNA polymerase sigma factor SigA
  • Template strand DNA
KeywordsTRANSCRIPTION/DNA/RNA / Cryo-EM / Synechocystis sp. PCC 6803 / Syn6803 / Transcription initiation / CTP-bound RPitc / Transcription / Cyanobacteria / TRANSCRIPTION-DNA-RNA complex
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-templated transcription / magnesium ion binding / DNA 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-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
RNA polymerase sigma factor, RpoD-like, cyanobacteria / DNA-directed RNA polymerase, subunit gamma / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / 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 ...RNA polymerase sigma factor, RpoD-like, cyanobacteria / DNA-directed RNA polymerase, subunit gamma / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / 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, 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 / 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-like DNA-binding domain superfamily
Similarity search - Domain/homology
CYTIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit gamma / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsYou, L.L. / Shen, L.Q. / Zhang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Chinese Academy of Sciences China
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2023
Title: Cryo-EM structure of Synechocystis sp. PCC 6803 CTP-bound RPitc
Authors: You, L.L. / Shen, L.Q. / Zhang, Y.
History
DepositionSep 27, 2022Deposition site: PDBJ / Processing site: RCSB
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit gamma
E: DNA-directed RNA polymerase subunit omega
F: RNA polymerase sigma factor SigA
Z: DNA-directed RNA polymerase subunit beta'
1: Nontemplate strand DNA
2: Template strand DNA
3: RNA (5'-R(*CP*CP*GP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)515,95115
Polymers515,28810
Non-polymers6635
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA-directed RNA polymerase subunit ... , 5 types, 6 molecules ABCDEZ

#1: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36657.344 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: rpoA, sll1818 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P73297, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 123719.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: rpoB, sll1787 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P77965, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit gamma / Polymerase / RNAP subunit gamma / RNA polymerase subunit gamma / Transcriptase subunit gamma


Mass: 71072.992 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: rpoC1, slr1265 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P74177, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 8749.776 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: rpoZ, ssl2982 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P74352, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 145371.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: rpoC2, sll1789 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P73334, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules 12

#7: DNA chain Nontemplate strand DNA


Mass: 20810.250 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain Template strand DNA


Mass: 20673.232 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules F3

#5: Protein RNA polymerase sigma factor SigA


Mass: 50335.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: sigA, rpoD, rpoD1, slr0653 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P74565
#9: RNA chain RNA (5'-R(*CP*CP*GP*A)-3')


Mass: 1239.818 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 5 molecules

#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#12: Chemical ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE / Cytidine triphosphate


Mass: 483.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O14P3

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The complex of Synechocystis sp. PCC 6803 CTP-bound RPitc
Type: COMPLEX / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.5 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Synechocystis sp. PCC 6803 (bacteria)1148
31synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2297

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Processing

EM software
IDNameVersionCategory
1RELION3.08particle selection
2EPUimage acquisition
4RELIONCTF correction
7UCSF Chimera10.14model fitting
9RELION3.08initial Euler assignment
10RELION3.08final Euler assignment
11RELION3.08classification
12RELION3.083D reconstruction
13PHENIX10.14model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 681334
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 145731 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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