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- PDB-8hhl: Cryo-EM structure of the Cas12m2-crRNA-target DNA full R-loop complex -

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Basic information

Entry
Database: PDB / ID: 8hhl
TitleCryo-EM structure of the Cas12m2-crRNA-target DNA full R-loop complex
Components
  • Cas12m2
  • NTS (36-MER)
  • TS (36-MER)
  • crRNA (56-MER)
KeywordsRNA BINDING PROTEIN / CRISPR-Cas / RNA BINDING PROTEIN-DNA COMPLEX
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesMycolicibacterium mucogenicum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsOmura, N.S. / Nakagawa, R. / Wu, Y.W. / Sudfeld, C. / Warren, V.R. / Hirano, H. / Kusakizako, T. / Kise, Y. / Lebbink, H.G.J. / Itoh, Y. ...Omura, N.S. / Nakagawa, R. / Wu, Y.W. / Sudfeld, C. / Warren, V.R. / Hirano, H. / Kusakizako, T. / Kise, Y. / Lebbink, H.G.J. / Itoh, Y. / Oost, V.D.J. / Nureki, O.
Funding support Japan, 1items
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, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 12, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 19, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Aug 9, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.3Aug 30, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: crRNA (56-MER)
C: TS (36-MER)
D: NTS (36-MER)
A: Cas12m2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)107,0298
Polymers106,8914
Non-polymers1384
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules CD

#2: DNA chain TS (36-MER)


Mass: 10976.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycolicibacterium mucogenicum (bacteria)
#3: DNA chain NTS (36-MER)


Mass: 11180.160 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycolicibacterium mucogenicum (bacteria)

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RNA chain / Protein , 2 types, 2 molecules BA

#1: RNA chain crRNA (56-MER)


Mass: 18277.932 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium mucogenicum (bacteria)
Production host: Escherichia coli (E. coli)
#4: Protein Cas12m2


Mass: 66457.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium mucogenicum (bacteria)
Production host: Escherichia coli (E. coli)

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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
1Cas12m2-crRNA-target DNACOMPLEX#1-#40MULTIPLE SOURCES
2crRNA-target DNACOMPLEX#1-#31MULTIPLE SOURCES
3Cas12m2COMPLEX#41RECOMBINANT
4crRNACOMPLEX#12RECOMBINANT
5target DNACOMPLEX#2-#32SYNTHETIC
Molecular weightExperimental value: NO
Source (natural)Organism: Mycolicibacterium mucogenicum (bacteria)
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
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: FEI TITAN 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: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 182116 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047339
ELECTRON MICROSCOPYf_angle_d0.48510470
ELECTRON MICROSCOPYf_dihedral_angle_d20.3611861
ELECTRON MICROSCOPYf_chiral_restr0.0351210
ELECTRON MICROSCOPYf_plane_restr0.004951

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