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- PDB-7c7l: Cryo-EM structure of the Cas12f1-sgRNA-target DNA complex -

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Basic information

Entry
Database: PDB / ID: 7c7l
TitleCryo-EM structure of the Cas12f1-sgRNA-target DNA complex
Components
  • (DNA (40-mer)) x 2
  • CRISPR-associated protein Cas14a.1
  • sgRNA
KeywordsRNA BINDING PROTEIN/RNA/DNA / Cas12f / Cas14 / sgRNA / target DNA / CRISPR / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
Transposase IS605, OrfB, C-terminal / Cas12f1-like, TNB domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endodeoxyribonuclease Cas12f1
Similarity search - Component
Biological speciesuncultured archaeon (environmental samples)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsTakeda, N.S. / Nakagawa, R. / Okazaki, S. / Hirano, H. / Kobayashi, K. / Kusakizako, T. / Nishizawa, T. / Yamashita, K. / Nishimasu, H. / Nureki, O.
CitationJournal: Mol Cell / Year: 2021
Title: Structure of the miniature type V-F CRISPR-Cas effector enzyme.
Authors: Satoru N Takeda / Ryoya Nakagawa / Sae Okazaki / Hisato Hirano / Kan Kobayashi / Tsukasa Kusakizako / Tomohiro Nishizawa / Keitaro Yamashita / Hiroshi Nishimasu / Osamu Nureki /
Abstract: RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also ...RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single- and double-stranded DNA targets. Here, we report the cryo-electron microscopy structure of Cas12f1 (also known as Cas14a) in complex with a guide RNA and its target DNA. Unexpectedly, the structure revealed that two Cas12f1 molecules assemble with the single guide RNA to recognize the double-stranded DNA target. Each Cas12f1 protomer adopts a different conformation and plays distinct roles in nucleic acid recognition and DNA cleavage, thereby explaining how the miniature Cas12f1 enzyme achieves RNA-guided DNA cleavage as an "asymmetric homodimer." Our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and provide a framework for the development of compact genome-engineering tools critical for therapeutic genome editing.
History
DepositionMay 26, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 23, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 30, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 17, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / refine
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: CRISPR-associated protein Cas14a.1
B: CRISPR-associated protein Cas14a.1
C: sgRNA
D: DNA (40-mer)
E: DNA (40-mer)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,4498
Polymers208,2535
Non-polymers1963
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21990 Å2
ΔGint-116 kcal/mol
Surface area57460 Å2

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Components

#1: Protein CRISPR-associated protein Cas14a.1 / Cas12f.1


Mass: 62748.598 Da / Num. of mol.: 2 / Mutation: D326A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) uncultured archaeon (environmental samples)
Production host: Escherichia coli (E. coli) / References: UniProt: A0A482D308
#2: RNA chain sgRNA


Mass: 58133.543 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) uncultured archaeon (environmental samples)
#3: DNA chain DNA (40-mer)


Mass: 12297.954 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (40-mer)


Mass: 12323.922 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of the Cas12f1-sgRNA-target DNA complexCOMPLEX#1-#40RECOMBINANT
2Cas12f1COMPLEX#11RECOMBINANT
3sgRNAORGANELLE OR CELLULAR COMPONENT#21RECOMBINANT
4target DNACOMPLEX#3-#41RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: uncultured archaeon (environmental samples)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
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: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 48.7 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0266 / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4RELION3CTF correction
5CTFFIND4.1.13CTF correction
11RELION3initial Euler assignment
12RELION3final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87253 / Symmetry type: POINT
RefinementResolution: 3.3→3.3 Å / Cor.coef. Fo:Fc: 0.731 / SU B: 22.846 / SU ML: 0.356 / Cross valid method: NONE / ESU R: 0.735
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.44518 --
obs0.44518 74091 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 101.179 Å2
Baniso -1Baniso -2Baniso -3
1--0.6 Å20.05 Å2-0.02 Å2
2--0.67 Å2-0.94 Å2
3----0.07 Å2
Refinement stepCycle: 1 / Total: 10148
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01210687
ELECTRON MICROSCOPYr_bond_other_d00.0188316
ELECTRON MICROSCOPYr_angle_refined_deg1.4981.49915154
ELECTRON MICROSCOPYr_angle_other_deg1.3851.86919232
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.5965862
ELECTRON MICROSCOPYr_dihedral_angle_2_deg37.33721.541331
ELECTRON MICROSCOPYr_dihedral_angle_3_deg20.774151228
ELECTRON MICROSCOPYr_dihedral_angle_4_deg18.1171544
ELECTRON MICROSCOPYr_chiral_restr0.0760.21531
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.029699
ELECTRON MICROSCOPYr_gen_planes_other0.0020.022490
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it8.8918.3393487
ELECTRON MICROSCOPYr_mcbond_other8.8818.3393486
ELECTRON MICROSCOPYr_mcangle_it14.23312.4764336
ELECTRON MICROSCOPYr_mcangle_other14.23412.4784337
ELECTRON MICROSCOPYr_scbond_it10.51613.1537200
ELECTRON MICROSCOPYr_scbond_other10.51613.157199
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other17.73219.72110819
ELECTRON MICROSCOPYr_long_range_B_refined37.93255141
ELECTRON MICROSCOPYr_long_range_B_other37.93255140
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3→3.078 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.2648 5499 -
obs--100 %

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