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- PDB-8wa1: The cryo-EM structure of the Nicotiana tabacum PEP-PAP-TEC2 -

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Basic information

Entry
Database: PDB / ID: 8wa1
TitleThe cryo-EM structure of the Nicotiana tabacum PEP-PAP-TEC2
Components
  • (DNA (24-mer)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • (Fructokinase-like ...) x 2
  • (Protein PLASTID TRANSCRIPTIONALLY ACTIVE ...) x 4
  • (superoxide dismutase) x 2
  • PAP1(pTAC3)
  • PAP13(pTAC18)
  • PAP8(pTAC6)
  • Pentatricopeptide repeat-containing protein At1g74850, chloroplastic-like
  • RNA (27-mer)
  • Thioredoxin-like protein CITRX1, chloroplastic
  • UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2, 6-diaminopimelate ligase-like
KeywordsTRANSCRIPTION / PEP / PAP
Function / homology
Function and homology information


etioplast organization / plastid transcription / positive regulation of red or far-red light signaling pathway / plastid-encoded plastid RNA polymerase complex / plastid organization / chloroplast nucleoid / acid-amino acid ligase activity / chloroplast organization / disulfide oxidoreductase activity / superoxide dismutase ...etioplast organization / plastid transcription / positive regulation of red or far-red light signaling pathway / plastid-encoded plastid RNA polymerase complex / plastid organization / chloroplast nucleoid / acid-amino acid ligase activity / chloroplast organization / disulfide oxidoreductase activity / superoxide dismutase / ligase activity / superoxide dismutase activity / protein-disulfide reductase activity / response to light stimulus / DNA-directed RNA polymerase complex / cell redox homeostasis / chloroplast / methyltransferase activity / ribonucleoside binding / DNA-directed RNA polymerase / kinase activity / DNA-directed RNA polymerase activity / regulation of cell shape / methylation / protein dimerization activity / cell division / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / mitochondrion / DNA binding / RNA binding / zinc ion binding / ATP binding / metal ion binding / nucleus
Similarity search - Function
Protein PLASTID TRANSCRIPTIONALLY ACTIVE 12 / Protein plastid transcriptionally active 7 / PLASTID TRANSCRIPTIONALLY ACTIVE protein 6 / Protein PLASTID TRANSCRIPTIONALLY ACTIVE 10 / Thioredoxin-like protein CITRX, chloroplastic / (S)-ureidoglycine aminohydrolase, cupin domain / EutQ-like cupin domain / : / PPR repeat / Pentacotripeptide-repeat region of PRORP ...Protein PLASTID TRANSCRIPTIONALLY ACTIVE 12 / Protein plastid transcriptionally active 7 / PLASTID TRANSCRIPTIONALLY ACTIVE protein 6 / Protein PLASTID TRANSCRIPTIONALLY ACTIVE 10 / Thioredoxin-like protein CITRX, chloroplastic / (S)-ureidoglycine aminohydrolase, cupin domain / EutQ-like cupin domain / : / PPR repeat / Pentacotripeptide-repeat region of PRORP / Pentacotripeptide-repeat region of PRORP / Smr domain / Smr domain profile. / UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / Rubisco LSMT, substrate-binding domain / Rubisco LSMT, substrate-binding domain superfamily / : / Rubisco LSMT substrate-binding / PPR repeat family / MurE/MurF, N-terminal / : / Mur ligase, N-terminal catalytic domain / Mur ligase family, catalytic domain / pfkB family of carbohydrate kinases signature 1. / Mur ligase, C-terminal / Mur ligase, C-terminal domain superfamily / Mur ligase, glutamate ligase domain / Mur ligase, central / Mur-like, catalytic domain superfamily / Mur ligase middle domain / Carbohydrate/purine kinase, PfkB, conserved site / Pentatricopeptide repeat domain / Manganese/iron superoxide dismutase, binding site / Manganese and iron superoxide dismutases signature. / Manganese/iron superoxide dismutase / Manganese/iron superoxide dismutase, N-terminal / Iron/manganese superoxide dismutases, alpha-hairpin domain / Pentatricopeptide (PPR) repeat profile. / Pentatricopeptide repeat / Manganese/iron superoxide dismutase, C-terminal / Manganese/iron superoxide dismutase, C-terminal domain superfamily / Manganese/iron superoxide dismutase, N-terminal domain superfamily / Iron/manganese superoxide dismutases, C-terminal domain / Carbohydrate kinase PfkB / pfkB family carbohydrate kinase / Ribokinase-like / Thioredoxin / SET domain / SET domain profile. / SET domain superfamily / SET domain / RmlC-like cupin domain superfamily / S1 domain profile. / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / Thioredoxin domain profile. / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / Thioredoxin domain / S1 domain / RmlC-like jelly roll fold / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 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 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase, RBP11-like subunit / 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 / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit gamma / Plastid transcriptionally active 6 / Protein PLASTID TRANSCRIPTIONALLY ACTIVE 7 isoform X1 / Thioredoxin-like protein CITRX1, chloroplastic / Protein PLASTID TRANSCRIPTIONALLY ACTIVE 12 ...: / DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit gamma / Plastid transcriptionally active 6 / Protein PLASTID TRANSCRIPTIONALLY ACTIVE 7 isoform X1 / Thioredoxin-like protein CITRX1, chloroplastic / Protein PLASTID TRANSCRIPTIONALLY ACTIVE 12 / (S)-ureidoglycine aminohydrolase cupin domain-containing protein / Protein PLASTID TRANSCRIPTIONALLY ACTIVE 10 / Pentatricopeptide repeat-containing protein At1g74850, chloroplastic / Fructokinase-like 2, chloroplastic / Fructokinase-like 1, chloroplastic / superoxide dismutase / superoxide dismutase / UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2,6-diaminopimelate ligase MurE homolog, chloroplastic / Protein PLASTID TRANSCRIPTIONALLY ACTIVE 14-like isoform X2 / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta''
Similarity search - Component
Biological speciesNicotiana tabacum (common tobacco)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsWu, X.X. / Zhang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Other governmentJCYJ-SHFY-2022-012 China
CitationJournal: Cell / Year: 2024
Title: Cryo-EM structures of the plant plastid-encoded RNA polymerase.
Authors: Xiao-Xian Wu / Wen-Hui Mu / Fan Li / Shu-Yi Sun / Chao-Jun Cui / Chanhong Kim / Fei Zhou / Yu Zhang /
Abstract: Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast ...Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.
History
DepositionSep 6, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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 beta
C: DNA-directed RNA polymerase subunit gamma
D: PAP1(pTAC3)
E: Pentatricopeptide repeat-containing protein At1g74850, chloroplastic-like
F: Protein PLASTID TRANSCRIPTIONALLY ACTIVE 10-like
G: superoxide dismutase
H: Protein PLASTID TRANSCRIPTIONALLY ACTIVE 12-like
I: Fructokinase-like 1, chloroplastic
J: Protein PLASTID TRANSCRIPTIONALLY ACTIVE 14-like isoform X2
K: PAP8(pTAC6)
L: superoxide dismutase
M: Thioredoxin-like protein CITRX1, chloroplastic
N: UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2, 6-diaminopimelate ligase-like
O: Protein PLASTID TRANSCRIPTIONALLY ACTIVE 7-like isoform X1
P: PAP13(pTAC18)
Q: DNA (24-mer)
R: DNA (24-mer)
S: RNA (27-mer)
a: DNA-directed RNA polymerase subunit alpha
c: DNA-directed RNA polymerase subunit beta''
i: Fructokinase-like 2, chloroplastic
m: Thioredoxin-like protein CITRX1, chloroplastic
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,249,62027
Polymers1,249,41923
Non-polymers2014
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: Protein DNA-directed RNA polymerase subunit alpha


Mass: 38663.543 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: P06269
#2: Protein DNA-directed RNA polymerase subunit beta


Mass: 120698.125 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: P06271
#3: Protein DNA-directed RNA polymerase subunit gamma


Mass: 79367.297 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A140G1Q3
#20: Protein DNA-directed RNA polymerase subunit beta''


Mass: 157057.734 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: P38550

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Protein , 8 types, 9 molecules DEGKLMmNP

#4: Protein PAP1(pTAC3)


Mass: 101413.672 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco)
#5: Protein Pentatricopeptide repeat-containing protein At1g74850, chloroplastic-like / PAP2(pTAC2)


Mass: 96666.289 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S3ZSI0
#7: Protein superoxide dismutase / PAP4(FSD3)


Mass: 30420.742 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S4CKK0
#11: Protein PAP8(pTAC6)


Mass: 38179.574 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S3X147
#12: Protein superoxide dismutase


Mass: 34825.203 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S4CIZ9
#13: Protein Thioredoxin-like protein CITRX1, chloroplastic


Mass: 20142.209 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S3YEW3
#14: Protein UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2, 6-diaminopimelate ligase-like / PAP11(MurE-like)


Mass: 85288.945 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S4D4H1
#16: Protein PAP13(pTAC18)


Mass: 17054.311 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S3YU01

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Protein PLASTID TRANSCRIPTIONALLY ACTIVE ... , 4 types, 4 molecules FHJO

#6: Protein Protein PLASTID TRANSCRIPTIONALLY ACTIVE 10-like / PAP3(pTAC10)


Mass: 80402.969 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S3YXM6
#8: Protein Protein PLASTID TRANSCRIPTIONALLY ACTIVE 12-like / PAP5(pTAC12)


Mass: 61281.289 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S3YPU3
#10: Protein Protein PLASTID TRANSCRIPTIONALLY ACTIVE 14-like isoform X2


Mass: 58183.145 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S4D7C7
#15: Protein Protein PLASTID TRANSCRIPTIONALLY ACTIVE 7-like isoform X1 / PAP12(pTAC7)


Mass: 19254.900 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S3XP85

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Fructokinase-like ... , 2 types, 2 molecules Ii

#9: Protein Fructokinase-like 1, chloroplastic / PAP6a(FLN1)


Mass: 55770.000 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S4CE74
#21: Protein Fructokinase-like 2, chloroplastic / PAP6b(FLN2)


Mass: 72534.133 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco) / References: UniProt: A0A1S4BFK7

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DNA chain , 2 types, 2 molecules QR

#17: DNA chain DNA (24-mer)


Mass: 7301.712 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#18: DNA chain DNA (24-mer)


Mass: 7386.807 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain , 1 types, 1 molecules S

#19: RNA chain RNA (27-mer)


Mass: 8720.280 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 4 molecules

#22: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#23: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#24: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe

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Details

Has ligand of interestN
Has protein modificationY

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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: Nicotiana tabacum PEP-PAP-TEC2 / Type: COMPLEX / Entity ID: #1-#3, #20, #4-#9, #21, #10-#19 / Source: MULTIPLE SOURCES
Source (natural)Organism: Nicotiana tabacum (common tobacco)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 57.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 88607 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 151.43 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003464174
ELECTRON MICROSCOPYf_angle_d0.626887301
ELECTRON MICROSCOPYf_chiral_restr0.04949787
ELECTRON MICROSCOPYf_plane_restr0.004211375
ELECTRON MICROSCOPYf_dihedral_angle_d12.58438350

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