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- EMDB-8996: CasX-gRNA-DNA(45bp) state I -

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Basic information

Entry
Database: EMDB / ID: EMD-8996
TitleCasX-gRNA-DNA(45bp) state I
Map dataCasX-gRNA-DNA(45bp) state I
Sample
  • Complex: CasX-gRNA-DNA(45bp) state I
    • Protein or peptide: CasX
    • DNA: DNA (35-MER)
    • DNA: DNA (35-MER)
    • RNA: RNA (112-MER)
Function / homologyTransposase
Function and homology information
Biological speciesDeltaproteobacteria (bacteria) / Deltaproteobacteria bacterium RIFCSPLOWO2_12_FULL_43_16 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLiu JJ / Orlova N / Nogales E / Doudna JA
CitationJournal: Nature / Year: 2019
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
DepositionJul 25, 2018-
Header (metadata) releaseJan 23, 2019-
Map releaseFeb 6, 2019-
UpdateFeb 27, 2019-
Current statusFeb 27, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6ny2
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8996.png

Downloads & links

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Map

FileDownload / File: emd_8996.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCasX-gRNA-DNA(45bp) state I
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 256 pix.
= 230.4 Å		0.9 Å/pix.
x 256 pix.
= 230.4 Å		0.9 Å/pix.
x 256 pix.
= 230.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-0.7965231 - 1.6927896
Average (Standard dev.)0.0024212748 (±0.058873024)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 230.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.90.90.9
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z230.400230.400230.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ364364364
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.7971.6930.002

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Supplemental data

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Sample components

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Entire : CasX-gRNA-DNA(45bp) state I

EntireName: CasX-gRNA-DNA(45bp) state I
Components
  • Complex: CasX-gRNA-DNA(45bp) state I
    • Protein or peptide: CasX
    • DNA: DNA (35-MER)
    • DNA: DNA (35-MER)
    • RNA: RNA (112-MER)

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Supramolecule #1: CasX-gRNA-DNA(45bp) state I

SupramoleculeName: CasX-gRNA-DNA(45bp) state I / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Deltaproteobacteria (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: CasX

MacromoleculeName: CasX / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Deltaproteobacteria bacterium RIFCSPLOWO2_12_FULL_43_16 (bacteria)
Molecular weightTheoretical: 113.054359 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: DNA (35-MER)

MacromoleculeName: DNA (35-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Deltaproteobacteria (bacteria)
Molecular weightTheoretical: 10.681896 KDa
SequenceString:
(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)

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Macromolecule #3: DNA (35-MER)

MacromoleculeName: DNA (35-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Deltaproteobacteria (bacteria)
Molecular weightTheoretical: 10.644836 KDa
SequenceString:
(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)

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Macromolecule #4: RNA (112-MER)

MacromoleculeName: RNA (112-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Deltaproteobacteria (bacteria)
Molecular weightTheoretical: 35.995398 KDa
SequenceString:
GCGCGUUUAU UCCAUCUUUG GAGCCAGUCC CAGCGACUAU GUCGUAUGGA CGAAGCGCUU AUUUAUCGGC CGAUAAGUAA AACGCAUCA AAGUGCUGCA GCAGAAAAUC AAA

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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
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 46.0 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

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 149361
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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