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- PDB-7v93: Cryo-EM structure of the Cas12c2-sgRNA binary complex -

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Basic information

Entry
Database: PDB / ID: 7v93
TitleCryo-EM structure of the Cas12c2-sgRNA binary complex
Components
  • cas12c2
  • sgRNASubgenomic mRNA
KeywordsRNA BINDING PROTEIN/RNA / cas12c / c2c3 / sgRNA / CRISPR / RNA binding protein-RNA complex
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesuncultured archaeon (environmental samples)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 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

Downloads & links

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Assembly

Deposited unit
A: cas12c2
B: sgRNA


Theoretical massNumber of molelcules
Total (without water)174,4962
Polymers174,4962
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6660 Å2
ΔGint-55 kcal/mol
Surface area49210 Å2

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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)

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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 complexCOMPLEXall0MULTIPLE SOURCES
2Cas12c2COMPLEX#11RECOMBINANT
3sgRNASubgenomic mRNACOMPLEX#21NATURAL
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: COPPER/RHODIUM / 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
Image recordingAverage exposure time: 5 sec. / Electron dose: 54 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
1RELION3particle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 289394 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: RECIPROCAL
RefinementResolution: 3→3 Å / Cor.coef. Fo:Fc: 0.908 / SU B: 13.97 / SU ML: 0.263 / ESU R: 0.428
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.39427 --
obs0.39427 97951 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 107.322 Å2
Refinement stepCycle: 1 / Total: 8792
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.010.0129112
ELECTRON MICROSCOPYr_bond_other_d0.030.0187847
ELECTRON MICROSCOPYr_angle_refined_deg1.4121.59512664
ELECTRON MICROSCOPYr_angle_other_deg1.6081.67618141
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.3915921
ELECTRON MICROSCOPYr_dihedral_angle_2_deg29.88622.433374
ELECTRON MICROSCOPYr_dihedral_angle_3_deg20.076151304
ELECTRON MICROSCOPYr_dihedral_angle_4_deg11.0821546
ELECTRON MICROSCOPYr_chiral_restr0.0820.21259
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.029198
ELECTRON MICROSCOPYr_gen_planes_other0.0030.022032
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_it12.53110.0913702
ELECTRON MICROSCOPYr_mcbond_other12.52710.093701
ELECTRON MICROSCOPYr_mcangle_it18.93515.1394617
ELECTRON MICROSCOPYr_mcangle_other18.93415.1414618
ELECTRON MICROSCOPYr_scbond_it13.65212.3145410
ELECTRON MICROSCOPYr_scbond_other13.65312.3095409
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other22.05518.0028048
ELECTRON MICROSCOPYr_long_range_B_refined40.424258.74746809
ELECTRON MICROSCOPYr_long_range_B_other40.424258.74746808
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.9→2.975 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.396 7167 -
obs--100 %

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