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- EMDB-21551: Cryo-EM structure of Cas12i-crRNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-21551
TitleCryo-EM structure of Cas12i-crRNA complex
Map datasharpened
Sample
  • Complex: Cryo-EM structure of CRISPR-Cas complex
    • Protein or peptide: Cas12i
    • RNA: crRNA
KeywordsCRISPR / HYDROLASE-DNA-RNA complex
Biological speciesLachnospiraceae bacterium ND2006 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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 16, 2020-
Header (metadata) releaseApr 15, 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: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6w62
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21551.png

Downloads & links

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Map

FileDownload / File: emd_21551.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 192 pix.
= 201.6 Å		1.05 Å/pix.
x 192 pix.
= 201.6 Å		1.05 Å/pix.
x 192 pix.
= 201.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.06969338 - 0.10533916
Average (Standard dev.)0.00025260614 (±0.0029071963)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 201.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z201.600201.600201.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0700.1050.000

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of CRISPR-Cas complex

EntireName: Cryo-EM structure of CRISPR-Cas complex
Components
  • Complex: Cryo-EM structure of CRISPR-Cas complex
    • Protein or peptide: Cas12i
    • RNA: crRNA

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Supramolecule #1: Cryo-EM structure of CRISPR-Cas complex

SupramoleculeName: Cryo-EM structure of 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: 125.879211 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 YRNGTMKDNR GNNIQIDFMK DFEAIADDET SLYYFNMKYC KLLQSSIRNH SSQAKEYREE IFELLRDGKL SVLKLSSLS NLSFVMFKVA KSLIGTYFGH LLKKPKNSKS DVKAPPITDE DKQKADPEMF ALRLALEEKR LNKVKSKKEV IANKIVAKAL ELRDKYGPV LIKGENISDT TKKGKKSSTN SFLMDWLARG VANKVKEMVM MHQGLEFVEV NPNFTSHQDP FVHKNPENTF R ARYSRCTP SELTEKNRKE ILSFLSDKPS KRPTNAYYNE GAMAFLATYG LKKNDVLGVS LEKFKQIMAN ILHQRSEDQL LF PSRGGMF YLATYKLDAD ATSVNWNGKQ FWVCNADLVA AYNVGLVDIQ KDFKKK

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Molecular weightTheoretical: 9.581721 KDa
SequenceString:
CAAAUUGUGC CCAUCGUUGG CACGCGUGCA

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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 recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 (6k x 4k) / #0 - Detector mode: SUPER-RESOLUTION / #0 - Average electron dose: 35.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Average electron dose: 1.3 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

Image recording ID1
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 122435
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING

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