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- EMDB-23601: Cryo-EM structure of CasPhi-2 (Cas12j) bound to crRNA and Phospho... -

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Basic information

Entry
Database: EMDB / ID: EMD-23601
TitleCryo-EM structure of CasPhi-2 (Cas12j) bound to crRNA and Phosphorothioate-DNA
Map dataLocSpiral map
Sample
  • Complex: Cryo-EM map of CasPhi bound to crRNA and phosphorothioate-DNA in the presence of the nuclease magnesium cofactor
    • Protein or peptide: CasPhi
    • RNA: crRNA
    • DNA: TS-DNA
    • DNA: NTS-DNA*
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsCRISPR / CasPhi / Cas12j / Nuclease / R-loop / crRNA / PAM / RNP / Complex / VIRAL PROTEIN-RNA-DNA / VIRAL PROTEIN-RNA-DNA complex
Biological speciesBiggievirus Mos11
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsPausch P / Soczek K
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Advancing Translational Sciences (NIH/NCATS)U01AI142817-02 United States
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: DNA interference states of the hypercompact CRISPR-CasΦ effector.
Authors: Patrick Pausch / Katarzyna M Soczek / Dominik A Herbst / Connor A Tsuchida / Basem Al-Shayeb / Jillian F Banfield / Eva Nogales / Jennifer A Doudna /
Abstract: CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and ...CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.
History
DepositionMar 8, 2021-
Header (metadata) releaseAug 4, 2021-
Map releaseAug 4, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lyt
  • Surface level: 8
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23601.png

Downloads & links

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Map

FileDownload / File: emd_23601.map.gz / Format: CCP4 / Size: 32.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocSpiral map
Voxel sizeX=Y=Z: 1.115 Å
Density
Contour LevelBy AUTHOR: 8.0 / Movie #1: 8
Minimum - Maximum0.0 - 23.719563999999998
Average (Standard dev.)0.29846948 (±1.0167133)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions204204204
Spacing204204204
CellA=B=C: 227.46 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1151.1151.115
M x/y/z204204204
origin x/y/z0.0000.0000.000
length x/y/z227.460227.460227.460
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS204204204
D min/max/mean0.00023.7200.298

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Supplemental data

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Mask #1

Fileemd_23601_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: cryoSPARC map

Fileemd_23601_additional_1.map
AnnotationcryoSPARC map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_23601_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_23601_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 : Cryo-EM map of CasPhi bound to crRNA and phosphorothioate-DNA in ...

EntireName: Cryo-EM map of CasPhi bound to crRNA and phosphorothioate-DNA in the presence of the nuclease magnesium cofactor
Components
  • Complex: Cryo-EM map of CasPhi bound to crRNA and phosphorothioate-DNA in the presence of the nuclease magnesium cofactor
    • Protein or peptide: CasPhi
    • RNA: crRNA
    • DNA: TS-DNA
    • DNA: NTS-DNA*
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Cryo-EM map of CasPhi bound to crRNA and phosphorothioate-DNA in ...

SupramoleculeName: Cryo-EM map of CasPhi bound to crRNA and phosphorothioate-DNA in the presence of the nuclease magnesium cofactor
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Biggievirus Mos11
Molecular weightTheoretical: 128 KDa

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Macromolecule #1: CasPhi

MacromoleculeName: CasPhi / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Biggievirus Mos11
Molecular weightTheoretical: 86.127188 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPKPAVESEF SKVLKKHFPG ERFRSSYMKR GGKILAAQGE EAVVAYLQGK SEEEPPNFQP PAKCHVVTKS RDFAEWPIMK ASEAIQRYI YALSTTERAA CKPGKSSESH AAWFAATGVS NHGYSHVQGL NLIFDHTLGR YDGVLKKVQL RNEKARARLE S INASRADE ...String:
MPKPAVESEF SKVLKKHFPG ERFRSSYMKR GGKILAAQGE EAVVAYLQGK SEEEPPNFQP PAKCHVVTKS RDFAEWPIMK ASEAIQRYI YALSTTERAA CKPGKSSESH AAWFAATGVS NHGYSHVQGL NLIFDHTLGR YDGVLKKVQL RNEKARARLE S INASRADE GLPEIKAEEE EVATNETGHL LQPPGINPSF YVYQTISPQA YRPRDEIVLP PEYAGYVRDP NAPIPLGVVR NR CDIQKGC PGYIPEWQRE AGTAISPKTG KAVTVPGLSP KKNKRMRRYW RSEKEKAQDA LLVTVRIGTD WVVIDVRGLL RNA RWRTIA PKDISLNALL DLFTGDPVID VRRNIVTFTY TLDACGTYAR KWTLKGKQTK ATLDKLTATQ TVALVAIDLG QTNP ISAGI SRVTQENGAL QCEPLDRFTL PDDLLKDISA YRIAWDRNEE ELRARSVEAL PEAQQAEVRA LDGVSKETAR TQLCA DFGL DPKRLPWDKM SSNTTFISEA LLSNSVSRDQ VFFTPAPKKG AKKKAPVEVM RKDRTWARAY KPRLSVEAQK LKNEAL WAL KRTSPEYLKL SRRKEELCRR SINYVIEKTR RRTQCQIVIP VIEDLNVRFF HGSGKRLPGW DNFFTAKKEN RWFIQGL HK AFSDLRTHRS FYVFEVRPER TSITCPKCGH CEVGNRDGEA FQCLSCGKTC NADLDVATHN LTQVALTGKT MPKREEPR D AQGTAPARKT KKASKSKAPP AEREDQTPAQ EPSQTSHHHH HH

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Biggievirus Mos11
Molecular weightTheoretical: 14.481651 KDa
SequenceString:
CAACGAUUGC CCCUCACGAG GGGACAGCUG GUAAUGGGAU ACCUU

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Macromolecule #3: TS-DNA

MacromoleculeName: TS-DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Biggievirus Mos11
Molecular weightTheoretical: 13.615714 KDa
SequenceString:
(DC)(DG)(DG)(DA)(DG)(DC)(DG)(DG)(DA)(DG) (DG)(DG)(DT)(DG)(DG)(DC)(DG)(DG)(DT)(DA) (DT)(DC)(DC)(DC)(DA)(DT)(DT)(DA)(DC) (DC)(DA)(DG)(DC)(DT)(DT)(DA)(DA)(DC)(DT) (DA) (DC)(DG)(DC)(DG)

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Macromolecule #4: NTS-DNA*

MacromoleculeName: NTS-DNA* / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Biggievirus Mos11
Molecular weightTheoretical: 13.072435 KDa
SequenceString:
(DC)(DG)(DC)(DG)(DT)(DA)(DG)(DT)(DT)(DA) (DT)(DC)(DG)(DA)(DC)(DC)(DA)(DT)(DT)(DA) (SC)(SC)(SC)(N)(N)(N)(DG)(DG)(DG) (DC)(DC)(DA)(DC)(DC)(DC)(DT)(DC)(DC)(DG) (DC) (DT)(DC)(DC)(DG)

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 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: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: NITROGEN / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: cryoSPARC ab initio 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: cryoSPARC (ver. 2.15) / Number images used: 410553
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
FSC plot (resolution estimation)

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