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- PDB-7v94: Cryo-EM structure of the Cas12c2-sgRNA-target DNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 7v94
TitleCryo-EM structure of the Cas12c2-sgRNA-target DNA ternary complex
Components
  • Cas12c2
  • sgRNASubgenomic mRNA
  • target DNA (non target strand)
  • target DNA (target strand)
KeywordsRNA BINDING PROTEIN/RNA/DNA / cas12c / c2c3 / sgRNA / target DNA / CRISPR / RNA binding protein-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesuncultured archaeon (environmental samples)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsKurihara, N. / Hirano, H. / Tomita, A. / Kobayashi, K. / Kusakizako, T. / Nishizawa, T. / Yamashita, K. / Nishimasu, H. / Nureki, O.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: Mol Cell / Year: 2022
Title: Structure of the type V-C CRISPR-Cas effector enzyme.
Authors: Nina Kurihara / Ryoya Nakagawa / Hisato Hirano / Sae Okazaki / Atsuhiro Tomita / Kan Kobayashi / Tsukasa Kusakizako / Tomohiro Nishizawa / Keitaro Yamashita / David A Scott / Hiroshi ...Authors: Nina Kurihara / Ryoya Nakagawa / Hisato Hirano / Sae Okazaki / Atsuhiro Tomita / Kan Kobayashi / Tsukasa Kusakizako / Tomohiro Nishizawa / Keitaro Yamashita / David A Scott / Hiroshi Nishimasu / Osamu Nureki /
Abstract: RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR ...RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) and recognizes double-stranded DNA targets with a short TN PAM. Here, we report the cryo-electron microscopy structures of the Cas12c2-guide RNA binary complex and the Cas12c2-guide RNA-target DNA ternary complex. The structures revealed that the crRNA and tracrRNA form an unexpected X-junction architecture, and that Cas12c2 recognizes a single T nucleotide in the PAM through specific hydrogen-bonding interactions with two arginine residues. Furthermore, our biochemical analyses indicated that Cas12c2 processes its precursor crRNA to a mature crRNA using the RuvC catalytic site through a unique mechanism. Collectively, our findings improve the mechanistic understanding of diverse type V CRISPR-Cas effectors.
History
DepositionAug 24, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 13, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 1, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jun 12, 2024Group: Data collection / Refinement description / Category: chem_comp_atom / chem_comp_bond / refine / Item: _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cas12c2
B: sgRNA
C: target DNA (target strand)
D: target DNA (non target strand)


Theoretical massNumber of molelcules
Total (without water)194,7954
Polymers194,7954
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cas12c2


Mass: 138348.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) uncultured archaeon (environmental samples)
Production host: Escherichia coli (E. coli)
#2: RNA chain sgRNA / Subgenomic mRNA


Mass: 36147.414 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) uncultured archaeon (environmental samples)
#3: DNA chain target DNA (target strand)


Mass: 10080.494 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain target DNA (non target strand)


Mass: 10218.596 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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
1Cas12c2-sgRNA-DNA complexCOMPLEXall0MULTIPLE SOURCES
2Cas12c2COMPLEX#11RECOMBINANT
3sgRNASubgenomic mRNACOMPLEX#21NATURAL
4DNACOMPLEX#3-#41NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11uncultured archaeon (environmental samples)115547
22uncultured archaeon (environmental samples)115547
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 FIELDBright-field microscopy / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingAverage exposure time: 2.6 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
7Buccaneermodel fitting
8Cootmodel fitting
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
14REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 534196 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 2.7→2.7 Å / Cor.coef. Fo:Fc: 0.898 / SU B: 9.107 / SU ML: 0.185 / ESU R: 0.258
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.34527 --
obs0.34527 174619 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 97.73 Å2
Refinement stepCycle: 1 / Total: 10839
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01211289
ELECTRON MICROSCOPYr_bond_other_d0.0290.0189428
ELECTRON MICROSCOPYr_angle_refined_deg1.4561.56815791
ELECTRON MICROSCOPYr_angle_other_deg1.4281.72621839
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.50551071
ELECTRON MICROSCOPYr_dihedral_angle_2_deg29.75222.603438
ELECTRON MICROSCOPYr_dihedral_angle_3_deg19.844151518
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.6071552
ELECTRON MICROSCOPYr_chiral_restr0.0760.21551
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.0211072
ELECTRON MICROSCOPYr_gen_planes_other0.0030.022508
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_it7.9038.8524299
ELECTRON MICROSCOPYr_mcbond_other7.9038.8524298
ELECTRON MICROSCOPYr_mcangle_it12.02413.3045365
ELECTRON MICROSCOPYr_mcangle_other12.02313.3045366
ELECTRON MICROSCOPYr_scbond_it8.57411.826990
ELECTRON MICROSCOPYr_scbond_other8.57511.8166989
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other14.07917.57310427
ELECTRON MICROSCOPYr_long_range_B_refined41.075293.36459831
ELECTRON MICROSCOPYr_long_range_B_other41.074293.36459832
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.6→2.668 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.964 12981 -
obs--100 %

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