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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-8996 | |||||||||
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Title | CasX-gRNA-DNA(45bp) state I | |||||||||
![]() | CasX-gRNA-DNA(45bp) state I | |||||||||
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Function / homology | Transposase![]() | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
![]() | Liu JJ / Orlova N / Nogales E / Doudna JA | |||||||||
![]() | ![]() Title: CasX enzymes comprise a distinct family of RNA-guided genome editors. Authors: Jun-Jie Liu / Natalia Orlova / Benjamin L Oakes / Enbo Ma / Hannah B Spinner / Katherine L M Baney / Jonathan Chuck / Dan Tan / Gavin J Knott / Lucas B Harrington / Basem Al-Shayeb / ...Authors: Jun-Jie Liu / Natalia Orlova / Benjamin L Oakes / Enbo Ma / Hannah B Spinner / Katherine L M Baney / Jonathan Chuck / Dan Tan / Gavin J Knott / Lucas B Harrington / Basem Al-Shayeb / Alexander Wagner / Julian Brötzmann / Brett T Staahl / Kian L Taylor / John Desmarais / Eva Nogales / Jennifer A Doudna / ![]() ![]() Abstract: The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of ...The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 59.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 12.9 KB 12.9 KB | Display Display | ![]() |
Images | ![]() | 108 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6ny2MC ![]() 8980C ![]() 8987C ![]() 8988C ![]() 8989C ![]() 8990C ![]() 8991C ![]() 8994C ![]() 6ny1C ![]() 6ny3C M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | CasX-gRNA-DNA(45bp) state I | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.9 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : CasX-gRNA-DNA(45bp) state I
Entire | Name: CasX-gRNA-DNA(45bp) state I |
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Components |
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-Supramolecule #1: CasX-gRNA-DNA(45bp) state I
Supramolecule | Name: CasX-gRNA-DNA(45bp) state I / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #1: CasX
Macromolecule | Name: CasX / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 113.054359 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MEKRINKIRK KLSADNATKP VSRSGPMKTL LVRVMTDDLK KRLEKRRKKP EVMPQVISNN AANNLRMLLD DYTKMKEAIL QVYWQEFKD DHVGLMCKFA QPASKKIDQN KLKPEMDEKG NLTTAGFACS QCGQPLFVYK LEQVSEKGKA YTNYFGRCNV A EHEKLILL ...String: MEKRINKIRK KLSADNATKP VSRSGPMKTL LVRVMTDDLK KRLEKRRKKP EVMPQVISNN AANNLRMLLD DYTKMKEAIL QVYWQEFKD DHVGLMCKFA QPASKKIDQN KLKPEMDEKG NLTTAGFACS QCGQPLFVYK LEQVSEKGKA YTNYFGRCNV A EHEKLILL AQLKPEKDSD EAVTYSLGKF GQRALDFYSI HVTKESTHPV KPLAQIAGNR YASGPVGKAL SDACMGTIAS FL SKYQDII IEHQKVVKGN QKRLESLREL AGKENLEYPS VTLPPQPHTK EGVDAYNEVI ARVRMWVNLN LWQKLKLSRD DAK PLLRLK GFPSFPVVER RENEVDWWNT INEVKKLIDA KRDMGRVFWS GVTAEKRNTI LEGYNYLPNE NDHKKREGSL ENPK KPAKR QFGDLLLYLE KKYAGDWGKV FDEAWERIDK KIAGLTSHIE REEARNAEDA QSKAVLTDWL RAKASFVLER LKEMD EKEF YACEIQLQKW YGDLRGNPFA VEAENRVVDI SGFSIGSDGH SIQYRNLLAW KYLENGKREF YLLMNYGKKG RIRFTD GTD IKKSGKWQGL LYGGGKAKVI DLTFDPDDEQ LIILPLAFGT RQGREFIWND LLSLETGLIK LANGRVIEKT IYNKKIG RD EPALFVALTF ERREVVDPSN IKPVNLIGVA RGENIPAVIA LTDPEGCPLP EFKDSSGGPT DILRIGEGYK EKQRAIQA A KEVEQRRAGG YSRKFASKSR NLADDMVRNS ARDLFYHAVT HDAVLVFANL SRGFGRQGKR TFMTERQYTK MEDWLTAKL AYEGLTSKTY LSKTLAQYTS KTCSNCGFTI TTADYDGMLV RLKKTSDGWA TTLNNKELKA EYQITYYNRY KRQTVEKELS AELDRLSEE SGNNDISKWT KGRRDEALFL LKKRFSHRPV QEQFVCLDCG HEVHAAEQAA LNIARSWLFL NSNSTEFKSY K SGKQPFVG AWQAFYKRRL KEVWKPNA |
-Macromolecule #2: DNA (35-MER)
Macromolecule | Name: DNA (35-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 10.681896 KDa |
Sequence | String: (DT)(DA)(DT)(DA)(DA)(DT)(DT)(DT)(DG)(DA) (DG)(DC)(DG)(DC)(DA)(DC)(DC)(DT)(DA)(DA) (DT)(DT)(DT)(DC)(DC)(DT)(DG)(DA)(DA) (DA)(DT)(DC)(DC)(DC)(DG) |
-Macromolecule #3: DNA (35-MER)
Macromolecule | Name: DNA (35-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 10.644836 KDa |
Sequence | String: (DC)(DG)(DG)(DG)(DA)(DT)(DT)(DT)(DC)(DA) (DT)(DC)(DC)(DT)(DG)(DC)(DA)(DG)(DC)(DA) (DT)(DC)(DC)(DC)(DC)(DG)(DA)(DC)(DC) (DC)(DG)(DT)(DA)(DT)(DA) |
-Macromolecule #4: RNA (112-MER)
Macromolecule | Name: RNA (112-MER) / type: rna / ID: 4 / Number of copies: 1 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 35.995398 KDa |
Sequence | String: GCGCGUUUAU UCCAUCUUUG GAGCCAGUCC CAGCGACUAU GUCGUAUGGA CGAAGCGCUU AUUUAUCGGC CGAUAAGUAA AACGCAUCA AAGUGCUGCA GCAGAAAAUC AAA |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Grid | Details: unspecified |
Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 46.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 149361 |
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Initial angle assignment | Type: RANDOM ASSIGNMENT |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |