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- EMDB-31808: Cryo-EM structure of the Cas12c2-sgRNA-target DNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-31808
TitleCryo-EM structure of the Cas12c2-sgRNA-target DNA ternary complex
Map datapostprocess_masked.mrc from Relion
Sample
  • Complex: Cas12c2-sgRNA-DNA complex
    • Complex: Cas12c2
      • Protein or peptide: Cas12c2
    • Complex: sgRNASubgenomic mRNA
      • RNA: sgRNASubgenomic mRNA
    • Complex: DNA
      • DNA: target DNA (target strand)
      • DNA: target DNA (non target strand)
Biological speciesuncultured archaeon (environmental samples) / synthetic construct (others)
Methodsingle 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, 1 items
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, 2021-
Header (metadata) releaseApr 13, 2022-
Map releaseApr 13, 2022-
UpdateJun 1, 2022-
Current statusJun 1, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_31808.png

Downloads & links

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Map

FileDownload / File: emd_31808.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpostprocess_masked.mrc from Relion
Voxel sizeX=Y=Z: 1.08073 Å
Density
Contour LevelBy AUTHOR: 0.045
Minimum - Maximum-0.27973133 - 0.3699717
Average (Standard dev.)0.00033910558 (±0.008525341)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 207.50015 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_31808_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Additional map: calculated by deepEMhancer using run class001.mrc from Refine3D

Fileemd_31808_additional_1.map
Annotationcalculated by deepEMhancer using run_class001.mrc from Refine3D
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: run half1 class001 unfil.mrc from Relion

Fileemd_31808_half_map_1.map
Annotationrun_half1_class001_unfil.mrc from Relion
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: run half2 class001 unfil.mrc from Relion

Fileemd_31808_half_map_2.map
Annotationrun_half2_class001_unfil.mrc from Relion
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Sample components

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Entire : Cas12c2-sgRNA-DNA complex

EntireName: Cas12c2-sgRNA-DNA complex
Components
  • Complex: Cas12c2-sgRNA-DNA complex
    • Complex: Cas12c2
      • Protein or peptide: Cas12c2
    • Complex: sgRNASubgenomic mRNA
      • RNA: sgRNASubgenomic mRNA
    • Complex: DNA
      • DNA: target DNA (target strand)
      • DNA: target DNA (non target strand)

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Supramolecule #1: Cas12c2-sgRNA-DNA complex

SupramoleculeName: Cas12c2-sgRNA-DNA complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: uncultured archaeon (environmental samples)

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Supramolecule #2: Cas12c2

SupramoleculeName: Cas12c2 / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: uncultured archaeon (environmental samples)

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Supramolecule #3: sgRNA

SupramoleculeName: sgRNA / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2

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Supramolecule #4: DNA

SupramoleculeName: DNA / type: complex / Chimera: Yes / ID: 4 / Parent: 1 / Macromolecule list: #3-#4

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Macromolecule #1: Cas12c2

MacromoleculeName: Cas12c2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: uncultured archaeon (environmental samples)
Molecular weightTheoretical: 138.348938 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKIEEGKGHH HHHHMTKHSI PLHAFRNSGA DARKWKGRIA LLAKRGKETM RTLQFPLEMS EPEAAAINTT PFAVAYNAIE GTGKGTLFD YWAKLHLAGF RFFPSGGAAT IFRQQAVFED ASWNAAFCQQ SGKDWPWLVP SKLYERFTKA PREVAKKDGS K KSIEFTQE ...String:
MKIEEGKGHH HHHHMTKHSI PLHAFRNSGA DARKWKGRIA LLAKRGKETM RTLQFPLEMS EPEAAAINTT PFAVAYNAIE GTGKGTLFD YWAKLHLAGF RFFPSGGAAT IFRQQAVFED ASWNAAFCQQ SGKDWPWLVP SKLYERFTKA PREVAKKDGS K KSIEFTQE NVANESHVSL VGASITDKTP EDQKEFFLKM AGALAEKFDS WKSANEDRIV AMKVIDEFLK SEGLHLPSLE NI AVKCSVE TKPDNATVAW HDAPMSGVQN LAIGVFATCA SRIDNIYDLN GGKLSKLIQE SATTPNVTAL SWLFGKGLEY FRT TDIDTI MQDFNIPASA KESIKPLVES AQAIPTMTVL GKKNYAPFRP NFGGKIDSWI ANYASRLMLL NDILEQIEPG FELP QALLD NETLMSGIDM TGDELKELIE AVYAWVDAAK QGLATLLGRG GNVDDAVQTF EQFSAMMDTL NGTLNTISAR YVRAV EMAG KDEARLEKLI ECKFDIPKWC KSVPKLVGIS GGLPKVEEEI KVMNAAFKDV RARMFVRFEE IAAYVASKGA GMDVYD ALE KRELEQIKKL KSAVPERAHI QAYRAVLHRI GRAVQNCSEK TKQLFSSKVI EMGVFKNPSH LNNFIFNQKG AIYRSPF DR SRHAPYQLHA DKLLKNDWLE LLAEISATLM ASESTEQMED ALRLERTRLQ LQLSGLPDWE YPASLAKPDI EVEIQTAL K MQLAKDTVTS DVLQRAFNLY SSVLSGLTFK LLRRSFSLKM RFSVADTTQL IYVPKVCDWA IPKQYLQAEG EIGIAARVV TESSPAKMVT EVEMKEPKAL GHFMQQAPHD WYFDASLGGT QVAGRIVEKG KEVGKERKLV GYRMRGNSAY KTVLDKSLVG NTELSQCSM IIEIPYTQTV DADFRAQVQA GLPKVSINLP VKETITASNK DEQMLFDRFV AIDLGERGLG YAVFDAKTLE L QESGHRPI KAITNLLNRT HHYEQRPNQR QKFQAKFNVN LSELRENTVG DVCHQINRIC AYYNAFPVLE YMVPDRLDKQ LK SVYESVT NRYIWSSTDA HKSARVQFWL GGETWEHPYL KSAKDKKPLV LSPGRGASGK GTSQTCSCCG RNPFDLIKDM KPR AKIAVV DGKAKLENSE LKLFERNLES KDDMLARRHR NERAGMEQPL TPGNYTVDEI KALLRANLRR APKNRRTKDT TVSE YHCVF SDCGKTMHAD ENAAVNIGGK FIADIEK

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: uncultured archaeon (environmental samples)
Molecular weightTheoretical: 36.147414 KDa
SequenceString:
GGAUACCACC CGUGCAUUUC UGGAUCAAUG AUCCGUACCU CAAUGUCCGG GCGCGCAGCU AGAGCGACCU GAAGAAAUUC AGGUUGGGU UUGAGGGGAA AUUAGGUGCG CUU

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Macromolecule #3: target DNA (target strand)

MacromoleculeName: target DNA (target strand) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.080494 KDa
SequenceString:
(DA)(DG)(DG)(DT)(DT)(DG)(DC)(DC)(DA)(DA) (DG)(DC)(DG)(DC)(DA)(DC)(DC)(DT)(DA)(DA) (DT)(DT)(DT)(DC)(DC)(DC)(DA)(DT)(DT) (DT)(DT)(DA)(DG)

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Macromolecule #4: target DNA (non target strand)

MacromoleculeName: target DNA (non target strand) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.218596 KDa
SequenceString:
(DC)(DT)(DA)(DA)(DA)(DA)(DT)(DG)(DG)(DG) (DA)(DA)(DA)(DT)(DT)(DA)(DG)(DG)(DT)(DG) (DC)(DG)(DC)(DT)(DT)(DG)(DG)(DC)(DA) (DA)(DC)(DC)(DT)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 2.6 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 534196
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL
Output model

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

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