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- EMDB-17757: Cryo-EM structure of the Cas12m-crRNA-target DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-17757
TitleCryo-EM structure of the Cas12m-crRNA-target DNA complex
Map dataMap sharpened using phenix.auto_sharpen_1.21rc1-4903 b_iso_to_d_cut procedure and 2.93A cutoff
Sample
  • Complex: Cas12m protein in complex with crRNA and cognate DNA, Gordonia otitidis NBRC 100426 system
    • Protein or peptide: Transposase
    • RNA: crRNA, chain B
    • DNA: DNA oligoduplex, target strand, chain C
    • DNA: DNA oligoduplex, non-target strand, chain D
KeywordsCas12 / Cas12m / CRISPR-Cas / RuvC / crRNA / PAM / RNA BINDING PROTEIN
Function / homologyTransposase
Function and homology information
Biological speciesGordonia otitidis NBRC 100426 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsSasnauskas G / Tamulaitiene G / Karvelis T / Bigelyte G / Siksnys V
Funding supportLithuania, 1 items
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, 2023-
Header (metadata) releaseFeb 7, 2024-
Map releaseFeb 7, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17757.png

Downloads & links

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Map

FileDownload / File: emd_17757.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap sharpened using phenix.auto_sharpen_1.21rc1-4903 b_iso_to_d_cut procedure and 2.93A cutoff
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 4.5
Minimum - Maximum-25.078455000000002 - 39.72278
Average (Standard dev.)0.000000000005109 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 220.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17757_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_17757_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17757_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17757_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas12m protein in complex with crRNA and cognate DNA, Gordonia ot...

EntireName: Cas12m protein in complex with crRNA and cognate DNA, Gordonia otitidis NBRC 100426 system
Components
  • Complex: Cas12m protein in complex with crRNA and cognate DNA, Gordonia otitidis NBRC 100426 system
    • Protein or peptide: Transposase
    • RNA: crRNA, chain B
    • DNA: DNA oligoduplex, target strand, chain C
    • DNA: DNA oligoduplex, non-target strand, chain D

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Supramolecule #1: Cas12m protein in complex with crRNA and cognate DNA, Gordonia ot...

SupramoleculeName: Cas12m protein in complex with crRNA and cognate DNA, Gordonia otitidis NBRC 100426 system
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Gordonia otitidis NBRC 100426 (bacteria)

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Macromolecule #1: Transposase

MacromoleculeName: Transposase / type: protein_or_peptide / ID: 1 / Details: Cas12m protein / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gordonia otitidis NBRC 100426 (bacteria)
Molecular weightTheoretical: 68.223586 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. DH10B (bacteria)
SequenceString: SNAGGGGMTR VTVQTAGVHY KWQMPDQLTQ QLRLAHDLRE DLVTLEYEYE DAVKAVWSSY PAVAALEAQV AELDERASEL ASTVKEEKS RQRTKRPSHP AVAQLAETRA QLKAAKASRR EAIASVRDEA TERLRTISDE RYAAQKQLYR DYCTDGLLYW A TFNAVLDH ...String:
SNAGGGGMTR VTVQTAGVHY KWQMPDQLTQ QLRLAHDLRE DLVTLEYEYE DAVKAVWSSY PAVAALEAQV AELDERASEL ASTVKEEKS RQRTKRPSHP AVAQLAETRA QLKAAKASRR EAIASVRDEA TERLRTISDE RYAAQKQLYR DYCTDGLLYW A TFNAVLDH HKTAVKRIAA HRKQGRAAQL RHHRWDGTGT ISVQLQRQAT DPARTPAIIA DADTGKWRSS LIVPWVNPDV WD TMDRASR RKAGRVVIRM RCGSSRNPDG TKTSEWIDVP VQQHRMLPAD ADITAAQLTV RREGADLRAT IGITAKIPDQ GEV DEGPTI AVHLGWRSSD HGTVVATWRS TEPLDIPETL RGVITTQSAE RTVGSIVVPH RIEQRVHHHA TVASHRDLAV DSIR DTLVA WLTEHGPQPH PYDGDPITAA SVQRWKAPRR FAWLALQWRD TPPPEGADIA ETLEAWRRAD KKLWLESEHG RGRAL RHRT DLHRQVAAYF AGVAGRIVVD DSDIAQIAGT AKHSELLTDV DRQIARRRAI AAPGMLRAAI VAAATRDEVP TTTVSH TGL SRVHAACGHE NPADDRYLMQ PVLCDGCGRT YDTDLSATIL MLQRASAATS N

UniProtKB: Transposase

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Macromolecule #2: crRNA, chain B

MacromoleculeName: crRNA, chain B / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Gordonia otitidis NBRC 100426 (bacteria)
Molecular weightTheoretical: 18.75124 KDa
SequenceString:
GGGUGUCAAC GCCAGCGCGG AGGCGUCAAA UCCGCGACAG UUGACCCAAC GUCGCCGG

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Macromolecule #3: DNA oligoduplex, target strand, chain C

MacromoleculeName: DNA oligoduplex, target strand, chain C / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.556727 KDa
SequenceString:
(DC)(DC)(DG)(DG)(DC)(DG)(DA)(DC)(DG)(DT) (DT)(DG)(DG)(DG)(DT)(DC)(DA)(DA)(DC)(DT) (DG)(DA)(DA)(DA)(DT)(DG)(DT)(DC)(DT) (DG)(DT)(DT)(DT)(DC)(DT)(DC)(DA)(DA)(DC) (DA) (DA)(DA)(DA)(DA)

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Macromolecule #4: DNA oligoduplex, non-target strand, chain D

MacromoleculeName: DNA oligoduplex, non-target strand, chain D / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.543744 KDa
SequenceString:
(DC)(DC)(DG)(DG)(DC)(DG)(DA)(DC)(DG)(DT) (DT)(DG)(DG)(DG)(DT)(DC)(DA)(DA)(DC)(DT) (DG)(DA)(DA)(DA)(DC)(DA)(DG)(DA)(DC) (DA)(DT)(DT)(DT)(DC)(DT)(DC)(DA)(DA)(DC) (DA) (DA)(DA)(DA)(DA)

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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: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
10.0 mMMgCl2magnesium chloride
40.0 mMTris-HClTris
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 92000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 2032 / Average exposure time: 46.33 sec. / Average electron dose: 29.7 e/Å2

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 204822
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

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

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