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- EMDB-21541: Cryo-EM structure of Cas12i(E894A)-crRNA-dsDNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-21541
TitleCryo-EM structure of Cas12i(E894A)-crRNA-dsDNA complex
Map dataCas12i-crRNA-dsDNA
Sample
  • Complex: CRISPR-Cas complex
    • Protein or peptide: Cas12i
    • DNA: TS
    • DNA: NTS
    • DNA: Substrate
    • RNA: crRNA
KeywordsCRISPR / HYDROLASE-DNA-RNA complex
Biological speciesLachnospiraceae bacterium ND2006 (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsChang L / Li Z / Zhang H
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Mechanisms for target recognition and cleavage by the Cas12i RNA-guided endonuclease.
Authors: Heng Zhang / Zhuang Li / Renjian Xiao / Leifu Chang /
Abstract: Cas12i is a recently identified type V CRISPR-Cas endonuclease that predominantly cleaves the non-target strand of a double-stranded DNA substrate. This nicking activity of Cas12i could potentially ...Cas12i is a recently identified type V CRISPR-Cas endonuclease that predominantly cleaves the non-target strand of a double-stranded DNA substrate. This nicking activity of Cas12i could potentially be used for genome editing with high specificity. To elucidate its mechanisms for target recognition and cleavage, we determined cryo-EM structures of Cas12i in multiple functional states. Cas12i pre-orders a seven-nucleotide seed sequence of the crRNA for target recognition and undergoes a two-step activation through crRNA-DNA hybridization. Formation of 14 base pairs activates the nickase activity, and 28-bp hybridization promotes cleavage of the target strand. The atomic structures and mechanistic insights gained should facilitate the manipulation of Cas12i for genome editing applications.
History
DepositionMar 13, 2020-
Header (metadata) releaseSep 16, 2020-
Map releaseSep 16, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0428
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0428
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6w5c
  • Surface level: 0.0428
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21541.png

Downloads & links

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Map

FileDownload / File: emd_21541.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas12i-crRNA-dsDNA
Voxel sizeX=Y=Z: 1.066 Å
Density
Contour LevelBy AUTHOR: 0.0428 / Movie #1: 0.0428
Minimum - Maximum-0.06751358 - 0.23422766
Average (Standard dev.)0.0017026073 (±0.009458656)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 149.23999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0661.0661.066
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z149.240149.240149.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.0680.2340.002

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Supplemental data

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Sample components

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Entire : CRISPR-Cas complex

EntireName: CRISPR-Cas complex
Components
  • Complex: CRISPR-Cas complex
    • Protein or peptide: Cas12i
    • DNA: TS
    • DNA: NTS
    • DNA: Substrate
    • RNA: crRNA

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Supramolecule #1: CRISPR-Cas complex

SupramoleculeName: CRISPR-Cas complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)

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Macromolecule #1: Cas12i

MacromoleculeName: Cas12i / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Molecular weightTheoretical: 125.722008 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSNKEKNASE TRKAYTTKMI PRSHDRMKLL GNFMDYLMDG TPIFFELWNQ FGGGIDRDII SGTANKDKIS DDLLLAVNWF KVMPINSKP QGVSPSNLAN LFQQYSGSEP DIQAQEYFAS NFDTEKHQWK DMRVEYERLL AELQLSRSDM HHDLKLMYKE K CIGLSLST ...String:
MSNKEKNASE TRKAYTTKMI PRSHDRMKLL GNFMDYLMDG TPIFFELWNQ FGGGIDRDII SGTANKDKIS DDLLLAVNWF KVMPINSKP QGVSPSNLAN LFQQYSGSEP DIQAQEYFAS NFDTEKHQWK DMRVEYERLL AELQLSRSDM HHDLKLMYKE K CIGLSLST AHYITSVMFG TGAKNNRQTK HQFYSKVIQL LEESTQINSV EQLASIILKA GDCDSYRKLR IRCSRKGATP SI LKIVQDY ELGTNHDDEV NVPSLIANLK EKLGRFEYEC EWKCMEKIKA FLASKVGPYY LGSYSAMLEN ALSPIKGMTT KNC KFVLKQ IDAKNDIKYE NEPFGKIVEG FFDSPYFESD TNVKWVLHPH HIGESNIKTL WEDLNAIHSK YEEDIASLSE DKKE KRIKV YQGDVCQTIN TYCEEVGKEA KTPLVQLLRY LYSRKDDIAV DKIIDGITFL SKKHKVEKQK INPVIQKYPS FNFGN NSKL LGKIISPKDK LKHNLKCNRN QVDNYIWIEI KVLNTKTMRW EKHHYALSST RFLEEVYYPA TSENPPDALA ARFRTK TNG YEGKPALSAE QIEQIRSAPV GLRKVKKRQM RLEAARQQNL LPRYTWGKDF NINICKRGNN FEVTLATKVK KKKEKNY KV VLGYDANIVR KNTYAAIEAH ANGDGVIDYN DLPVKPIESG FVTVESQVRD KSYDQLSYNG VKLLYCKPHV ESRRSFLE K YNGTMKDNRG NNIQIDFMKD FEAIADDETS LYYFNMKYCK LLQSSIRNHS SQAKEYREEI FELLRDGKLS VLKLSSLSN LSFVMFKVAK SLIGTYFGHL LKKPKNSKSD VKAPPITDED KQKADPEMFA LRLALEEKRL NKVKSKKEVI ANKIVAKALE LRDKYGPVL IKGENISDTT KKGKKSSTNS FLMDWLARGV ANKVKEMVMM HQGLEFVEVN PNFTSHQDPF VHKNPENTFR A RYSRCTPS ELTEKNRKEI LSFLSDKPSK RPTNAYYNEG AMAFLATYGL KKNDVLGVSL EKFKQIMANI LHQRSEDQLL FP SRGGMFY LATYKLDADA TSVNWNGKQF WVCNADLVAA YNVGLVDIQK DFKKK

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Macromolecule #2: TS

MacromoleculeName: TS / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Molecular weightTheoretical: 11.083159 KDa
SequenceString:
(DC)(DA)(DG)(DA)(DC)(DC)(DA)(DT)(DC)(DG) (DA)(DA)(DG)(DC)(DA)(DA)(DT)(DC)(DC)(DA) (DG)(DC)(DA)(DC)(DG)(DC)(DG)(DA)(DA) (DA)(DG)(DG)(DA)(DC)(DT)(DG)

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Macromolecule #3: NTS

MacromoleculeName: NTS / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Molecular weightTheoretical: 2.970954 KDa
SequenceString:
(DC)(DA)(DG)(DT)(DC)(DC)(DT)(DT)(DT)(DC)

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Macromolecule #4: Substrate

MacromoleculeName: Substrate / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Molecular weightTheoretical: 2.788862 KDa
SequenceString:
(DA)(DA)(DA)(DG)(DG)(DA)(DC)(DT)(DG)

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Macromolecule #5: crRNA

MacromoleculeName: crRNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 18.859102 KDa
SequenceString:
ACGCAAACAA UUUUUGUGCC CAUCGUUGGC ACGCGUGCUG GAUUGCUUCG AUGGUCUGC

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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

Concentration.5 mg/mL
BufferpH: 7.5
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 35.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 731231
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING

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