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- EMDB-61040: Cryo-EM structure of CasLambda2-crRNA-target DNA ternary complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-61040
TitleCryo-EM structure of CasLambda2-crRNA-target DNA ternary complex in the NTS-cleaving state
Map data
Sample
  • Complex: CasLambda2-crRNA-target DNA ternary complex
    • Protein or peptide: Cas Lambda2
    • RNA: RNA (58-MER)
    • DNA: DNA (40-MER)
    • DNA: DNA (40-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-THIO-MONOPHOSPHATE
  • Ligand: 2-DEOXY-CYTIDINE-5'-THIOPHOSPHORATE
  • Ligand: 2-DEOXY-ADENOSINE -5'-THIO-MONOPHOSPHATE
KeywordsCRISPR-CAS12 / GENOME ENGINEERING / HYDROLASE-RNA-DNA COMPLEX / DNA BINDING PROTEIN
Biological speciesunidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsOmura SN / Hirano H / Itoh Y / Nureki O
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Commun Biol / Year: 2025
Title: Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2.
Authors: Satoshi N Omura / Lauren E Alfonse / Alexa Ornstein / Hayato Morinaga / Hisato Hirano / Yuzuru Itoh / Gabrielle Munoz / Anthony J Garrity / Gregory R Hoffman / Tia DiTommaso / Winston X Yan ...Authors: Satoshi N Omura / Lauren E Alfonse / Alexa Ornstein / Hayato Morinaga / Hisato Hirano / Yuzuru Itoh / Gabrielle Munoz / Anthony J Garrity / Gregory R Hoffman / Tia DiTommaso / Winston X Yan / David R Cheng / David A Scott / Zachary Maben / Osamu Nureki /
Abstract: RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ-also referred to as Cas12n-is a recently identified ...RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ-also referred to as Cas12n-is a recently identified miniature type V nuclease encoded in phage genomes. Given its demonstrated nuclease activity in both mammalian and plant cells, Casλ has emerged as a promising candidate for genome-editing applications. However, the precise molecular mechanisms of Casλ family enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.
History
DepositionAug 1, 2024-
Header (metadata) releaseJun 11, 2025-
Map releaseJun 11, 2025-
UpdateJun 25, 2025-
Current statusJun 25, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61040.png

Downloads & links

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Map

FileDownload / File: emd_61040.map.gz / Format: CCP4 / Size: 6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 116 pix.
= 144.42 Å		1.25 Å/pix.
x 116 pix.
= 144.42 Å		1.25 Å/pix.
x 116 pix.
= 144.42 Å

Surface

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Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.245 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.55
Minimum - Maximum-2.8148324 - 5.795751
Average (Standard dev.)0.015080507 (±0.20775743)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin626262
Dimensions116116116
Spacing116116116
CellA=B=C: 144.42 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_61040_msk_1.map
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Half map: #2

Fileemd_61040_half_map_1.map
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Half map: #1

Fileemd_61040_half_map_2.map
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Sample components

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Entire : CasLambda2-crRNA-target DNA ternary complex

EntireName: CasLambda2-crRNA-target DNA ternary complex
Components
  • Complex: CasLambda2-crRNA-target DNA ternary complex
    • Protein or peptide: Cas Lambda2
    • RNA: RNA (58-MER)
    • DNA: DNA (40-MER)
    • DNA: DNA (40-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-THIO-MONOPHOSPHATE
  • Ligand: 2-DEOXY-CYTIDINE-5'-THIOPHOSPHORATE
  • Ligand: 2-DEOXY-ADENOSINE -5'-THIO-MONOPHOSPHATE

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Supramolecule #1: CasLambda2-crRNA-target DNA ternary complex

SupramoleculeName: CasLambda2-crRNA-target DNA ternary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: unidentified (others)

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Macromolecule #1: Cas Lambda2

MacromoleculeName: Cas Lambda2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 88.746492 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHGG MKKSIKFKVK GNCPITKDVI NEYKEYYNKC SDWIKNNLTS ITIGEMAKFL QETLGKDVAY ISMGLSDEWK DKPLYHLFT KKYHTNNADN LLYYYIKEKN LDGYKGNTLN IGNTFFRQFG YFKLVVSNYR TKIRTLNCEI KRKKIDADST S EDIEMQTM ...String:
MGHHHHHHGG MKKSIKFKVK GNCPITKDVI NEYKEYYNKC SDWIKNNLTS ITIGEMAKFL QETLGKDVAY ISMGLSDEWK DKPLYHLFT KKYHTNNADN LLYYYIKEKN LDGYKGNTLN IGNTFFRQFG YFKLVVSNYR TKIRTLNCEI KRKKIDADST S EDIEMQTM YEIIKHNLNK KTDWDEFISY IENVENPNID NINRYKLLRK CFCENENMIK NKLELLSIEQ LKNFGGCIMK QH INSMTLI IQHFKIEEKE NSLGFILNLP LNKKQYQIEL WGNRQVNKGT KERDAFLNTY GENIVFIINN DELYVVFSYE YEL EKEEAN FVKTVGLDVN FKHAFFVTSE KDNCHLDGYI NLYKYLLEHD EFTNLLTNDE KKDYEELSKV VTFCPFENQL LFAR YNKMS KFCKKEQVLS KLLYALQKQL KDENRTKEYI YVSCVNKLRA KYVSYFILKE KYYEKQKEYD IEMGFVDDST ESKES MDKR RTEFPFRNTP VANELLSKLN NVQQDINGCL KNIINYIYKI FEQNGYKIVA LENLENSNFE KKQVLPTIKS LLKYHK LEN QNVNDIKASD KVKEYIENGY YELITNENNE IVDAKYTEKG AMKVKNANFF NLMMKSLHFA SVKDEFVLLS NNGKTQI AL VPSEFTSQMD STDHCLYMKK NDKGKLVKAD KKEVRTKQEK HINGLNADFN AANNIKYIVE NEVWREIFCT RPKKAEYN V PSLDTTKKGP SAILHMLKKI EAIKILETEK

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Macromolecule #2: RNA (58-MER)

MacromoleculeName: RNA (58-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 18.692006 KDa
SequenceString:
GGCUUGUUGU AUAUAUUCUU UUAUAGGUAU UAAACAACGG AGAUGAGGUG CGCGUGGC

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Macromolecule #3: DNA (40-MER)

MacromoleculeName: DNA (40-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 12.591749 KDa
SequenceString:
(DT)(DG)(DT)(DC)(DT)(DA)(DT)(DT)(DT)(DA) (DG)(DG)(DA)(DG)(DA)(DT)(DG)(DA)(DG)(DG) (DT)(DG)(DC)(DG)(DC)(DG)(DT)(DG)(GS) (SC)(AS)(SC)(SC)(AS)(PST)(SC)(AS)(PST)(AS) (PST)

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Macromolecule #4: DNA (40-MER)

MacromoleculeName: DNA (40-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 12.418669 KDa
SequenceString:
(AS)(PST)(AS)(PST)(GS)(AS)(PST)(GS)(GS)(PST) (GS)(SC)(DC)(DA)(DC)(DG)(DC)(DG)(DC) (DA)(DC)(DC)(DT)(DC)(DA)(DT)(DC)(DT)(DC) (DC)(DT)(DA)(DA)(DA)(DT)(DA)(DG)(DA)(DC) (DA)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: GUANOSINE-5'-THIO-MONOPHOSPHATE

MacromoleculeName: GUANOSINE-5'-THIO-MONOPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: GS
Molecular weightTheoretical: 363.287 Da
Chemical component information

ChemComp-GS:
GUANOSINE-5'-THIO-MONOPHOSPHATE

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Macromolecule #7: 2-DEOXY-CYTIDINE-5'-THIOPHOSPHORATE

MacromoleculeName: 2-DEOXY-CYTIDINE-5'-THIOPHOSPHORATE / type: ligand / ID: 7 / Number of copies: 3 / Formula: SC
Molecular weightTheoretical: 323.263 Da
Chemical component information

ChemComp-SC:
2-DEOXY-CYTIDINE-5'-THIOPHOSPHORATE

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Macromolecule #8: 2-DEOXY-ADENOSINE -5'-THIO-MONOPHOSPHATE

MacromoleculeName: 2-DEOXY-ADENOSINE -5'-THIO-MONOPHOSPHATE / type: ligand / ID: 8 / Number of copies: 2 / Formula: AS
Molecular weightTheoretical: 347.287 Da
Chemical component information

ChemComp-AS:
2-DEOXY-ADENOSINE -5'-THIO-MONOPHOSPHATE

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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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 97017
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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