+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23600 | |||||||||
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Title | Cryo-EM structure of CasPhi-2 (Cas12j) bound to crRNA and DNA | |||||||||
Map data | LocSpiral map | |||||||||
Sample |
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Keywords | CRISPR / CasPhi / Cas12j / Nuclease / R-loop / crRNA / PAM / RNP / Complex / VIRAL PROTEIN-RNA-DNA complex | |||||||||
Biological species | Biggievirus Mos11 / Phage #D (virus) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.05 Å | |||||||||
Authors | Pausch P / Soczek K | |||||||||
Funding support | United States, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_23600.map.gz | 882.6 KB | EMDB map data format | |
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Header (meta data) | emd-23600-v30.xml emd-23600.xml | 22.2 KB 22.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_23600_fsc.xml | 7.6 KB | Display | FSC data file |
Images | emd_23600.png | 56.8 KB | ||
Masks | emd_23600_msk_1.map | 32.4 MB | Mask map | |
Filedesc metadata | emd-23600.cif.gz | 6.4 KB | ||
Others | emd_23600_additional_1.map.gz emd_23600_additional_2.map.gz emd_23600_half_map_1.map.gz emd_23600_half_map_2.map.gz | 30.5 MB 16.2 MB 30.1 MB 30.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23600 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23600 | HTTPS FTP |
-Validation report
Summary document | emd_23600_validation.pdf.gz | 887.5 KB | Display | EMDB validaton report |
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Full document | emd_23600_full_validation.pdf.gz | 887 KB | Display | |
Data in XML | emd_23600_validation.xml.gz | 14.5 KB | Display | |
Data in CIF | emd_23600_validation.cif.gz | 18.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23600 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23600 | HTTPS FTP |
-Related structure data
Related structure data | 7lysMC 7lytC 7m5oC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_23600.map.gz / Format: CCP4 / Size: 32.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | LocSpiral map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.115 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_23600_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: cryoSPARC sharp map
File | emd_23600_additional_1.map | ||||||||||||
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Annotation | cryoSPARC sharp map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: cryoSPARC map
File | emd_23600_additional_2.map | ||||||||||||
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Annotation | cryoSPARC map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_23600_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_23600_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Cryo-EM map of CasPhi bound to crRNA and DNA
Entire | Name: Cryo-EM map of CasPhi bound to crRNA and DNA |
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Components |
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-Supramolecule #1: Cryo-EM map of CasPhi bound to crRNA and DNA
Supramolecule | Name: Cryo-EM map of CasPhi bound to crRNA and DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Biggievirus Mos11 |
Molecular weight | Theoretical: 124 KDa |
-Macromolecule #1: CasPhi-2
Macromolecule | Name: CasPhi-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Biggievirus Mos11 |
Molecular weight | Theoretical: 86.127188 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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 |
-Macromolecule #2: crRNA
Macromolecule | Name: crRNA / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: Phage #D (virus) |
Molecular weight | Theoretical: 14.481651 KDa |
Sequence | String: CAACGAUUGC CCCUCACGAG GGGACAGCUG GUAAUGGGAU ACCUU |
-Macromolecule #3: TS-DNA
Macromolecule | Name: TS-DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Phage #D (virus) |
Molecular weight | Theoretical: 12.033733 KDa |
Sequence | String: (DC)(DG)(DG)(DA)(DG)(DG)(DG)(DT)(DG)(DA) (DA)(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) |
-Macromolecule #4: NTS-DNA
Macromolecule | Name: NTS-DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Phage #D (virus) |
Molecular weight | Theoretical: 11.864624 KDa |
Sequence | String: (DC)(DG)(DC)(DG)(DT)(DA)(DG)(DT)(DT)(DA) (DT)(DC)(DG)(DA)(DC)(DC)(DA)(DT)(DT)(DA) (DC)(DC)(DC)(DT)(DA)(DT)(DG)(DG)(DA) (DA)(DC)(DA)(DC)(DC)(DC)(DT)(DC)(DC)(DG) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Grid | Model: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: NITROGEN / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |