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- PDB-9mku: FnoCas12a bridge helix variant state 2 -

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Basic information

Entry
Database: PDB / ID: 9mku
TitleFnoCas12a bridge helix variant state 2
Components
  • CRISPR-associated endonuclease Cas12a
  • TS DNA
  • crRNA
KeywordsDNA BINDING PROTEIN / Cas12a / bridge helix / loop-to-helical transition / conformational cascade / allostery / off-target DNA cleavage
Function / homology
Function and homology information


Bacillus subtilis ribonuclease / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding
Similarity search - Function
: / CRISPR-associated endonuclease Cpf1 PI domain / : / CRISPR-associated endonuclease Cpf1 REC2 domain / CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas12a
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida U112 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsGanguly, C. / Thomas, L.M. / Aribam, S.D. / Martin, L. / Rajan, R.
Funding support United States, 3items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MCB-1716423 United States
National Science Foundation (NSF, United States)MCB-2424888 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30GM145423 United States
CitationJournal: Nat Commun / Year: 2026
Title: Bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation.
Authors: Chhandosee Ganguly / Swarmistha Devi Aribam / Alberto Monteiro Dos Santos / Lindsie Martin / Leonard M Thomas / Yihan Shao / Rakhi Rajan /
Abstract: CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella ...CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12a) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH). In this work, we use a combinatorial approach to understand the molecular mechanisms of BH-mediated activation of Cas12a for DNA cleavage. We report five structures of FnoCas12a that are at different states of conformational activation. Comparison of the variant and wild-type (FnoCas12a) structures, along with activity assays and computational simulations, establishes the loop-to-helical transition and bending of the BH as an allosteric trigger for RNA-DNA hybrid propagation. These changes track with the previously reported coupled remodeling of BH and helix 1 of RuvC motif-II as well as the REC lobe movements needed to accommodate the growing hybrid. The transition of the BH is essential for the loop-to-helical transition of the "lid", which in turn opens the RuvC active site pocket for DNA entry and cleavage. Pairwise 3D structural comparison of the BH and RuvC of Cas12 and Cas9 families provides insight into the diversity of BH's structural organization in these mechanistically similar enzymes.
History
DepositionDec 18, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 11, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: crRNA
C: TS DNA
A: CRISPR-associated endonuclease Cas12a


Theoretical massNumber of molelcules
Total (without water)173,5673
Polymers173,5673
Non-polymers00
Water00
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

#1: RNA chain crRNA


Mass: 13749.146 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain TS DNA


Mass: 7302.752 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Protein CRISPR-associated endonuclease Cas12a / CRISPR-associated endonuclease Cpf1 / FnCas12a / FnCpf1


Mass: 152515.516 Da / Num. of mol.: 1 / Mutation: K969P, D970P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Francisella tularensis subsp. novicida U112 (bacteria)
Gene: cas12a, cpf1, FTN_1397 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0Q7Q2, deoxyribonuclease I, Bacillus subtilis ribonuclease
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: Ternary complex of FnoCRISPR-Cas12a / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.152 MDa / Experimental value: NO
Source (natural)Organism: Francisella tularensis subsp. novicida U112 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 4 K

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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: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 1.25 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286 / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67352 / Symmetry type: POINT
RefinementHighest resolution: 4 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211016
ELECTRON MICROSCOPYf_angle_d0.5114960
ELECTRON MICROSCOPYf_dihedral_angle_d11.9971757
ELECTRON MICROSCOPYf_chiral_restr0.0381630
ELECTRON MICROSCOPYf_plane_restr0.0031786

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