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- PDB-8qma: Structure of the plastid-encoded RNA polymerase complex (PEP) fro... -

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Basic information

Entry
Database: PDB / ID: 8qma
TitleStructure of the plastid-encoded RNA polymerase complex (PEP) from Sinapis alba
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • FLN2
  • PAP1
  • PAP10
  • PAP11
  • PAP12 (DNA-directed RNA polymerase subunit omega)
  • PAP3
  • PAP4
  • PAP5
  • PAP6
  • PAP7
  • PAP8
  • PAP9
  • PTAC18
KeywordsTRANSCRIPTION / Chloroplasts / Gene Expression / RNA / Polymerase
Function / homology
Function and homology information


chloroplast / DNA-directed RNA polymerase complex / 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
Similarity search - Function
DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / 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 Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / DNA-directed RNA polymerase, subunit beta-prime ...DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / 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 Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, domain 2 superfamily / 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, 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
Similarity search - Domain/homology
: / S-ADENOSYLMETHIONINE / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta''
Similarity search - Component
Biological speciesSinapis alba (white mustard)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
Authorsdo Prado, P.F.V. / Ahrens, F.M. / Pfannschmidt, T. / Hillen, H.S.
Funding support Germany, 5items
OrganizationGrant numberCountry
German Research Foundation (DFG)FOR2848 Germany
German Research Foundation (DFG)SFB1190 Germany
German Research Foundation (DFG)SFB1565 Germany
German Research Foundation (DFG)EXC 2067/1 390729940 Germany
German Research Foundation (DFG)PF323-7 Germany
CitationJournal: Mol Cell / Year: 2024
Title: Structure of the multi-subunit chloroplast RNA polymerase.
Authors: Paula F V do Prado / Frederik M Ahrens / Monique Liebers / Noah Ditz / Hans-Peter Braun / Thomas Pfannschmidt / Hauke S Hillen /
Abstract: Chloroplasts contain a dedicated genome that encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase ...Chloroplasts contain a dedicated genome that encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. Here, we report the cryo-EM structure of a 19-subunit PEP complex from Sinapis alba (white mustard). The structure reveals that the PEP core resembles prokaryotic and nuclear RNAPs but contains chloroplast-specific features that mediate interactions with the PAPs. The PAPs are unrelated to known transcription factors and arrange around the core in a unique fashion. Their structures suggest potential functions during transcription in the chemical environment of chloroplasts. These results reveal structural insights into chloroplast transcription and provide a framework for understanding photosynthesis gene expression.
History
DepositionSep 21, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 20, 2024Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: PAP4
H: PAP5
K: PAP8
L: PAP9
M: PAP10
N: PAP10
O: PAP11
P: PAP12 (DNA-directed RNA polymerase subunit omega)
R: PTAC18
S: PAP6
A: DNA-directed RNA polymerase subunit beta
B: DNA-directed RNA polymerase subunit beta''
C: DNA-directed RNA polymerase subunit alpha
D: DNA-directed RNA polymerase subunit alpha
E: PAP1
F: PAP3
I: FLN2
J: PAP7
T: DNA-directed RNA polymerase subunit beta'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,117,94923
Polymers1,117,37319
Non-polymers5764
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 13 types, 14 molecules GHKLMNOPRSEFIJ

#1: Protein PAP4


Mass: 30404.408 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#2: Protein PAP5


Mass: 60884.246 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#3: Protein PAP8


Mass: 38039.504 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#4: Protein PAP9


Mass: 34008.883 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#5: Protein PAP10


Mass: 20851.234 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#6: Protein PAP11


Mass: 85061.648 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#7: Protein PAP12 (DNA-directed RNA polymerase subunit omega)


Mass: 18835.195 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#8: Protein PTAC18


Mass: 16430.916 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#9: Protein PAP6


Mass: 52435.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#13: Protein PAP1


Mass: 103467.867 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#14: Protein PAP3


Mass: 79815.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#15: Protein FLN2


Mass: 68527.102 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
#16: Protein PAP7


Mass: 55675.461 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDT

#10: Protein DNA-directed RNA polymerase subunit beta / Polymerase / PEP / Plastid-encoded RNA polymerase subunit beta / RNA polymerase subunit beta


Mass: 121163.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
References: UniProt: A0A6C0M5W1, DNA-directed RNA polymerase
#11: Protein DNA-directed RNA polymerase subunit beta'' / Polymerase


Mass: 156388.719 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard) / References: UniProt: A0A6C0M829
#12: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / PEP / Plastid-encoded RNA polymerase subunit alpha / RNA polymerase subunit alpha


Mass: 37885.652 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
References: UniProt: A0A6C0M610, DNA-directed RNA polymerase
#17: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / PEP / Plastid-encoded RNA polymerase subunit beta' / RNA polymerase subunit beta'


Mass: 78761.867 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sinapis alba (white mustard)
References: UniProt: A0A6C0M5W0, DNA-directed RNA polymerase

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Non-polymers , 3 types, 4 molecules

#18: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#19: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#20: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE / S-Adenosyl methionine


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Plastid-encoded DNA-dependent RNA polymerase (PEP) / Type: COMPLEX / Entity ID: #1-#17 / Source: NATURAL
Molecular weightValue: 1 MDa / Experimental value: NO
Source (natural)Organism: Sinapis alba (white mustard)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris1
20.1 mMEDTAEthylenediaminetetraacetic acid1
310 mMMagnesium chlorideMgCl21
40.1 mMSodium fluorideNaF1
50.1 mMPMSF1
61 mM2-mercapto ethanol1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.7 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123874 / Algorithm: BACK PROJECTION / Symmetry type: POINT

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