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- PDB-9izr: Cryo-EM structure of CasLambda2-crRNA-target DNA ternary complex ... -

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Basic information

Entry
Database: PDB / ID: 9izr
TitleCryo-EM structure of CasLambda2-crRNA-target DNA ternary complex in the NTS-cleaving state
Components
  • (DNA (40-MER)) x 2
  • Cas Lambda2
  • RNA (58-MER)
KeywordsDNA BINDING PROTEIN / CRISPR-CAS12 / GENOME ENGINEERING / HYDROLASE-RNA-DNA COMPLEX
Function / homology2-DEOXY-ADENOSINE -5'-THIO-MONOPHOSPHATE / GUANOSINE-5'-THIO-MONOPHOSPHATE / 2-DEOXY-CYTIDINE-5'-THIOPHOSPHORATE / DNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesunidentified (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsOmura, S.N. / Hirano, H. / Itoh, Y. / Nureki, O.
Funding support Japan, 2items
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, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 11, 2025Provider: repository / Type: Initial release
Revision 1.0Jun 11, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 11, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 11, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 11, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 11, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 11, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Jun 11, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 25, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas Lambda2
B: RNA (58-MER)
C: DNA (40-MER)
D: DNA (40-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,67118
Polymers132,4494
Non-polymers2,22214
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain DNA (40-MER)


Mass: 12591.749 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli)
#4: DNA chain DNA (40-MER)


Mass: 12418.669 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein Cas Lambda2


Mass: 88746.492 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli)
#2: RNA chain RNA (58-MER)


Mass: 18692.006 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 4 types, 14 molecules

#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-GS / GUANOSINE-5'-THIO-MONOPHOSPHATE


Type: DNA linking / Mass: 363.287 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O6PS
#7: Chemical ChemComp-SC / 2-DEOXY-CYTIDINE-5'-THIOPHOSPHORATE


Type: DNA linking / Mass: 323.263 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C9H14N3O6PS
#8: Chemical ChemComp-AS / 2-DEOXY-ADENOSINE -5'-THIO-MONOPHOSPHATE


Type: DNA linking / Mass: 347.287 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H14N5O5PS

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Details

Has ligand of interestN
Has protein modificationN

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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

ComponentName: CasLambda2-crRNA-target DNA ternary complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: unidentified (others)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21_5207: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97017 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048544
ELECTRON MICROSCOPYf_angle_d0.51911926
ELECTRON MICROSCOPYf_dihedral_angle_d17.1753682
ELECTRON MICROSCOPYf_chiral_restr0.0391350
ELECTRON MICROSCOPYf_plane_restr0.0031192

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