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- EMDB-34803: Cryo-EM structure of the Cas12m2-crRNA-target DNA full R-loop complex -

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Basic information

Entry
Database: EMDB / ID: EMD-34803
TitleCryo-EM structure of the Cas12m2-crRNA-target DNA full R-loop complex
Map data
Sample
  • Complex: Cas12m2-crRNA-target DNA
    • Complex: crRNA-target DNA
      • Complex: crRNA
        • RNA: crRNA (56-MER)
      • Complex: target DNA
        • DNA: TS (36-MER)
        • DNA: NTS (36-MER)
    • Complex: Cas12m2
      • Protein or peptide: Cas12m2
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
KeywordsCRISPR-Cas / RNA BINDING PROTEIN-DNA COMPLEX / RNA BINDING PROTEIN
Biological speciesMycolicibacterium mucogenicum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsOmura NS / Nakagawa R / Wu YW / Sudfeld C / Warren VR / Hirano H / Kusakizako T / Kise Y / Lebbink HGJ / Itoh Y ...Omura NS / Nakagawa R / Wu YW / Sudfeld C / Warren VR / Hirano H / Kusakizako T / Kise Y / Lebbink HGJ / Itoh Y / Oost VDJ / Nureki O
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Mechanistic and evolutionary insights into a type V-M CRISPR-Cas effector enzyme.
Authors: Satoshi N Omura / Ryoya Nakagawa / Christian Südfeld / Ricardo Villegas Warren / Wen Y Wu / Hisato Hirano / Charlie Laffeber / Tsukasa Kusakizako / Yoshiaki Kise / Joyce H G Lebbink / ...Authors: Satoshi N Omura / Ryoya Nakagawa / Christian Südfeld / Ricardo Villegas Warren / Wen Y Wu / Hisato Hirano / Charlie Laffeber / Tsukasa Kusakizako / Yoshiaki Kise / Joyce H G Lebbink / Yuzuru Itoh / John van der Oost / Osamu Nureki /
Abstract: RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR- ...RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.
History
DepositionNov 16, 2022-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34803.png

Downloads & links

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Map

FileDownload / File: emd_34803.map.gz / Format: CCP4 / Size: 4.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.16 Å/pix.
x 110 pix.
= 127.82 Å		1.16 Å/pix.
x 100 pix.
= 116.2 Å		1.16 Å/pix.
x 108 pix.
= 125.496 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.162 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.65
Minimum - Maximum-2.448977 - 5.075161
Average (Standard dev.)-0.012179287 (±0.25256017)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin534442
Dimensions100108110
Spacing108100110
CellA: 125.495995 Å / B: 116.2 Å / C: 127.81999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_34803_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34803_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34803_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas12m2-crRNA-target DNA

EntireName: Cas12m2-crRNA-target DNA
Components
  • Complex: Cas12m2-crRNA-target DNA
    • Complex: crRNA-target DNA
      • Complex: crRNA
        • RNA: crRNA (56-MER)
      • Complex: target DNA
        • DNA: TS (36-MER)
        • DNA: NTS (36-MER)
    • Complex: Cas12m2
      • Protein or peptide: Cas12m2
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Cas12m2-crRNA-target DNA

SupramoleculeName: Cas12m2-crRNA-target DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Mycolicibacterium mucogenicum (bacteria)

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Supramolecule #2: crRNA-target DNA

SupramoleculeName: crRNA-target DNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3

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Supramolecule #3: Cas12m2

SupramoleculeName: Cas12m2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4

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Supramolecule #4: crRNA

SupramoleculeName: crRNA / type: complex / ID: 4 / Parent: 2 / Macromolecule list: #1

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Supramolecule #5: target DNA

SupramoleculeName: target DNA / type: complex / ID: 5 / Parent: 2 / Macromolecule list: #2-#3

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Macromolecule #1: crRNA (56-MER)

MacromoleculeName: crRNA (56-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Mycolicibacterium mucogenicum (bacteria)
Molecular weightTheoretical: 18.277932 KDa
SequenceString:
GUGUCAUAGC CCAGCUUGGC GGGCGAAGGC CAAGACGGAG AUGAGGUGCG CGUGGC

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Macromolecule #2: TS (36-MER)

MacromoleculeName: TS (36-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycolicibacterium mucogenicum (bacteria)
Molecular weightTheoretical: 10.976068 KDa
SequenceString:
(DG)(DA)(DT)(DG)(DG)(DT)(DG)(DC)(DC)(DA) (DC)(DG)(DC)(DG)(DC)(DA)(DC)(DC)(DT)(DC) (DA)(DT)(DC)(DT)(DC)(DC)(DC)(DA)(DA) (DA)(DT)(DA)(DG)(DA)(DC)(DA)

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Macromolecule #3: NTS (36-MER)

MacromoleculeName: NTS (36-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycolicibacterium mucogenicum (bacteria)
Molecular weightTheoretical: 11.18016 KDa
SequenceString:
(DT)(DG)(DT)(DC)(DT)(DA)(DT)(DT)(DT)(DG) (DG)(DG)(DA)(DG)(DA)(DT)(DG)(DA)(DG)(DG) (DT)(DG)(DC)(DG)(DC)(DG)(DT)(DG)(DG) (DC)(DA)(DC)(DC)(DA)(DT)(DC)

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Macromolecule #4: Cas12m2

MacromoleculeName: Cas12m2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium mucogenicum (bacteria)
Molecular weightTheoretical: 66.457 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GGMTTMTVHT MGVHYKWQIP EVLRQQLWLA HNLREDLVSL QLAYDDDLKA IWSSYPDVAQ AEDTMAAAEA DAVALSERVK QARIEARSK KISTELTQQL RDAKKRLKDA RQARRDAIAV VKDDAAERRK ARSDQLAADQ KALYGQYCRD GDLYWASFNT V LDHHKTAV ...String:
GGMTTMTVHT MGVHYKWQIP EVLRQQLWLA HNLREDLVSL QLAYDDDLKA IWSSYPDVAQ AEDTMAAAEA DAVALSERVK QARIEARSK KISTELTQQL RDAKKRLKDA RQARRDAIAV VKDDAAERRK ARSDQLAADQ KALYGQYCRD GDLYWASFNT V LDHHKTAV KRIAAQRASG KPATLRHHRF DGSGTIAVQL QRQAGAPPRT PMVLADEAGK YRNVLHIPGW TDPDVWEQMT RS QCRQSGR VTVRMRCGST DGQPQWIDLP VQVHRWLPAD ADITGAELVV TRVAGIYRAK LCVTARIGDT EPVTSGPTVA LHL GWRSTE EGTAVATWRS DAPLDIPFGL RTVMRVDAAG TSGIIVVPAT IERRLTRTEN IASSRSLALD ALRDKVVGWL SDND APTYR DAPLEAATVK QWKSPQRFAS LAHAWKDNGT EISDILWAWF SLDRKQWAQQ ENGRRKALGH RDDLYRQIAA VISDQ AGHV LVDDTSVAEL SARAMERTEL PTEVQQKIDR RRDHAAPGGL RASVVAAMTR DGVPVTIVAA ADFTRTHSRC GHVNPA DDR YLSNPVRCDG CGAMYDQDRS FVTLMLRAAT APSNP

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 182116
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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