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- PDB-9n9c: Cryo-EM structure of the dCas12f-gRNA complex -

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Basic information

Entry
Database: PDB / ID: 9n9c
TitleCryo-EM structure of the dCas12f-gRNA complex
Components
  • Nuclease-deactivated Cas12f
  • gRNA (88-MER)
KeywordsRNA BINDING PROTEIN/RNA / dCas12f / binary complex / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesFlagellimonas taeanensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsChang, L. / Sternberg, S.H. / Xiao, R. / Hoffmann, F.T. / Wiegand, T. / Xie, D.
Funding support United States, 2items
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
CitationJournal: To Be Published
Title: Structural basis of RNA-guided transcription via Cas12f-Sigma factor association
Authors: Renjian, X. / Leifu, C.
History
DepositionFeb 10, 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

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Assembly

Deposited unit
A: Nuclease-deactivated Cas12f
B: Nuclease-deactivated Cas12f
c: gRNA (88-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,7744
Polymers115,7493
Non-polymers241
Water00
1


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

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Components

#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: RNA chain gRNA (88-MER)


Mass: 29922.770 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Flagellimonas taeanensis (bacteria)
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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 complex / Type: COMPLEX / Entity ID: #1-#2 / 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: BRIGHT 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: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 116049 / Symmetry type: POINT
RefinementHighest resolution: 3.28 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0057935
ELECTRON MICROSCOPYf_angle_d0.60111081
ELECTRON MICROSCOPYf_dihedral_angle_d17.7892036
ELECTRON MICROSCOPYf_chiral_restr0.041284
ELECTRON MICROSCOPYf_plane_restr0.0051057

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