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- PDB-9n9q: Cryo-EM structure of the RNA-guided transcription complex -

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Basic information

Entry
Database: PDB / ID: 9n9q
TitleCryo-EM structure of the RNA-guided transcription complex
Components
  • (DNA-directed RNA polymerase subunit ...) x 3
  • DNA non-template strand
  • DNA template strand
  • Nuclease-deactivated Cas12f
  • RNA polymerase Rpb6
  • RpoE
  • gRNA (85-MER)
KeywordsRNA BINDING PROTEIN/DNA/RNA / dCas12f / partial R-Loop / RNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
DNA-directed RNA polymerase, subunit beta-prime, bacterial type / 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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 ...DNA-directed RNA polymerase, subunit beta-prime, bacterial type / 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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / 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 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, RBP11-like subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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
DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit alpha / RNA polymerase Rpb6 / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesFlagellimonas taeanensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsChang, L. / Sternberg, S.H. / Xiao, R. / Hoffmann, F.T. / Wiegand, T. / Xie, D.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM138675 United States
National Science Foundation (NSF, United States)2339799 United States
National Science Foundation (NSF, United States)2239685 United States
The Pew Charitable Trusts United States
CitationJournal: To Be Published
Title: Structural basis of RNA-guided transcription via Cas12f-Sigma factor association
Authors: Renjian, X. / Leifu, C.
History
DepositionFeb 11, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 24, 2025Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nuclease-deactivated Cas12f
B: Nuclease-deactivated Cas12f
C: RpoE
D: DNA-directed RNA polymerase subunit alpha
E: DNA-directed RNA polymerase subunit alpha
F: DNA-directed RNA polymerase subunit beta
G: DNA-directed RNA polymerase subunit beta'
H: RNA polymerase Rpb6
a: DNA non-template strand
b: DNA template strand
c: gRNA (85-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)597,97613
Polymers597,88611
Non-polymers902
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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Protein , 3 types, 4 molecules ABCH

#1: Protein Nuclease-deactivated Cas12f


Mass: 42913.262 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Flagellimonas taeanensis (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein RpoE


Mass: 31011.486 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Flagellimonas taeanensis (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#6: Protein RNA polymerase Rpb6


Mass: 12565.037 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Flagellimonas taeanensis (bacteria) / Gene: SAMN05216293_3853 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1M7BX03

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DNA-directed RNA polymerase subunit ... , 3 types, 4 molecules DEFG

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


Mass: 37228.574 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Flagellimonas taeanensis (bacteria) / Gene: rpoA, SAMN05216293_2985 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A1M6YWR9, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 142961.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Flagellimonas taeanensis (bacteria) / Gene: rpoB, SAMN05216293_2127 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A3A1NXR2, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 160063.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Flagellimonas taeanensis (bacteria) / Gene: rpoC, SAMN05216293_2128 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A3A1NXQ1, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules ab

#7: DNA chain DNA non-template strand


Mass: 30316.391 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Flagellimonas taeanensis (bacteria)
#8: DNA chain DNA template strand


Mass: 30761.699 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Flagellimonas taeanensis (bacteria)

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RNA chain , 1 types, 1 molecules c

#9: RNA chain gRNA (85-MER)


Mass: 29922.770 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Flagellimonas taeanensis (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)

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Non-polymers , 2 types, 2 molecules

#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestY
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: dCas12f-gRNA-DNA-RpoE-RNAP complex / Type: COMPLEX / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Source (natural)Organism: Flagellimonas taeanensis (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.6
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 59.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 54193 / Symmetry type: POINT
RefinementHighest resolution: 2.88 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00339096
ELECTRON MICROSCOPYf_angle_d0.53653587
ELECTRON MICROSCOPYf_dihedral_angle_d16.8757131
ELECTRON MICROSCOPYf_chiral_restr0.0426077
ELECTRON MICROSCOPYf_plane_restr0.0046189

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