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- PDB-8pm4: Cryo-EM structure of the Cas12m-crRNA-target DNA complex -

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Basic information

Entry
Database: PDB / ID: 8pm4
TitleCryo-EM structure of the Cas12m-crRNA-target DNA complex
Components
  • DNA oligoduplex, non-target strand, chain D
  • DNA oligoduplex, target strand, chain C
  • Transposase
  • crRNA, chain B
KeywordsRNA BINDING PROTEIN / Cas12 / Cas12m / CRISPR-Cas / RuvC / crRNA / PAM
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / Transposase
Function and homology information
Biological speciesGordonia otitidis NBRC 100426 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsSasnauskas, G. / Tamulaitiene, G. / Karvelis, T. / Bigelyte, G. / Siksnys, V.
Funding supportLithuania, 1items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-21-8Lithuania
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.
Authors: Greta Bigelyte / Brigita Duchovska / Rimante Zedaveinyte / Giedrius Sasnauskas / Tomas Sinkunas / Indre Dalgediene / Giedre Tamulaitiene / Arunas Silanskas / Darius Kazlauskas / Lukas ...Authors: Greta Bigelyte / Brigita Duchovska / Rimante Zedaveinyte / Giedrius Sasnauskas / Tomas Sinkunas / Indre Dalgediene / Giedre Tamulaitiene / Arunas Silanskas / Darius Kazlauskas / Lukas Valančauskas / Julene Madariaga-Marcos / Ralf Seidel / Virginijus Siksnys / Tautvydas Karvelis /
Abstract: Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) ...Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) effector proteins and show that they provide protection against bacteriophages and plasmids through the targeted DNA binding rather than DNA cleavage. Biochemical assays suggest that Cas12m effectors can act as roadblocks inhibiting DNA transcription and/or replication, thereby triggering interference against invaders. Cryo-EM structure of Gordonia otitidis (Go) Cas12m ternary complex provided here reveals the structural mechanism of DNA binding ensuring interference. Harnessing GoCas12m innate ability to bind DNA target we fused it with adenine deaminase TadA-8e and showed an efficient A-to-G editing in Escherichia coli and human cells. Overall, this study expands our understanding of the functionally diverse Cas12 protein family, revealing DNA-binding dependent interference mechanism of Cas12m effectors that could be harnessed for engineering of compact base-editing tools.
History
DepositionJun 28, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Structure summary / Category: entity / entity_name_com / Item: _entity.details
Revision 1.2Apr 24, 2024Group: Database references / Category: 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
A: Transposase
B: crRNA, chain B
C: DNA oligoduplex, target strand, chain C
D: DNA oligoduplex, non-target strand, chain D


Theoretical massNumber of molelcules
Total (without water)114,0754
Polymers114,0754
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Transposase / / Cas12m protein


Mass: 68223.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Cas12m protein
Source: (gene. exp.) Gordonia otitidis NBRC 100426 (bacteria)
Gene: GOOTI_202_00040
Production host: Escherichia coli str. K-12 substr. DH10B (bacteria)
References: UniProt: H5TRP0
#2: RNA chain crRNA, chain B


Mass: 18751.240 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Gordonia otitidis NBRC 100426 (bacteria)
#3: DNA chain DNA oligoduplex, target strand, chain C


Mass: 13556.727 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA oligoduplex, non-target strand, chain D


Mass: 13543.744 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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: Cas12m protein in complex with crRNA and cognate DNA, Gordonia otitidis NBRC 100426 system
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Gordonia otitidis NBRC 100426 (bacteria)
Source (recombinant)Organism: Escherichia coli str. K-12 substr. DH10B (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
210 mMmagnesium chlorideMgCl21
340 mMTris-HClTris1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal magnification: 92000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 46.33 sec. / Electron dose: 29.7 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2032

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
2EPU3.2image acquisition
7UCSF ChimeraX1.3model fitting
8Coot0.9.8.1model fitting
13cryoSPARC4.2.13D reconstruction
14PHENIX1.21rc1_4903model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204822 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 66.78 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00726963
ELECTRON MICROSCOPYf_angle_d0.6349913
ELECTRON MICROSCOPYf_chiral_restr0.04421172
ELECTRON MICROSCOPYf_plane_restr0.0055929
ELECTRON MICROSCOPYf_dihedral_angle_d17.64472752

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