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- PDB-8hhl: Cryo-EM structure of the Cas12m2-crRNA-target DNA full R-loop complex -
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Open data
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Basic information
Entry | Database: PDB / ID: 8hhl | ||||||
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Title | Cryo-EM structure of the Cas12m2-crRNA-target DNA full R-loop complex | ||||||
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![]() | RNA BINDING PROTEIN / CRISPR-Cas / RNA BINDING PROTEIN-DNA COMPLEX | ||||||
Function / homology | DNA / DNA (> 10) / RNA / RNA (> 10)![]() | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.87 Å | ||||||
![]() | 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. ...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 | ![]()
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![]() | ![]() 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. | ||||||
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 191.6 KB | Display | ![]() |
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PDB format | ![]() | 141.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.1 MB | Display | |
Data in XML | ![]() | 28.1 KB | Display | |
Data in CIF | ![]() | 42.7 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 34803MC ![]() 8hhmC ![]() 8hioC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
-DNA chain , 2 types, 2 molecules CD
#2: DNA chain | Mass: 10976.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
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#3: DNA chain | Mass: 11180.160 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
-RNA chain / Protein , 2 types, 2 molecules BA
#1: RNA chain | Mass: 18277.932 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Production host: ![]() ![]() |
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#4: Protein | Mass: 66457.000 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Production host: ![]() ![]() |
-Non-polymers , 2 types, 4 molecules ![](data/chem/img/MG.gif)
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#5: Chemical | #6: Chemical | ChemComp-ZN / | |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Molecular weight | Experimental value: NO | ||||||||||||||||||||||||||||||||||||
Source (natural) | Organism: ![]() | ||||||||||||||||||||||||||||||||||||
Source (recombinant) |
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Buffer solution | pH: 7.6 | ||||||||||||||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 182116 / Symmetry type: POINT | ||||||||||||||||||||||||
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