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- PDB-6ny2: CasX-gRNA-DNA(45bp) state I -

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Basic information

Entry
Database: PDB / ID: 6ny2
TitleCasX-gRNA-DNA(45bp) state I
Components
  • CasX
  • DNA Non-target strand
  • DNA target strand
  • RNA (110-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / CasX / sgRNA / target DNA / CRISPR / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Uncharacterized protein
Function and homology information
Biological speciesDeltaproteobacteria bacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLiu, J.J. / Orlova, N. / Nogales, E. / Doudna, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)p01 United States
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
DepositionFeb 10, 2019Deposition site: RCSB / Processing site: RCSB
SupersessionFeb 27, 2019ID: 6E79
Revision 1.0Feb 27, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Dec 25, 2019Group: Database references / Category: database_2

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Structure visualization

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Assembly

Deposited unit
C: DNA target strand
D: DNA Non-target strand
Y: CasX
B: RNA (110-MER)


Theoretical massNumber of molelcules
Total (without water)174,9324
Polymers174,9324
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21450 Å2
ΔGint-140 kcal/mol
Surface area66590 Å2

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Components

#1: DNA chain DNA target strand


Mass: 13823.874 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Deltaproteobacteria bacterium (bacteria)
#2: DNA chain DNA Non-target strand


Mass: 13742.822 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Deltaproteobacteria bacterium (bacteria)
#3: Protein CasX


Mass: 108092.836 Da / Num. of mol.: 1 / Mutation: D672A, E769A, D935A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deltaproteobacteria bacterium (bacteria)
Gene: DD725_10130 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A357BT59
#4: RNA chain RNA (110-MER)


Mass: 39272.391 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Deltaproteobacteria bacterium (bacteria)
Sequence detailsThe CasX construct has the following sequence: ...The CasX construct has the following sequence: SNAMEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQKWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITTADYDGMLVRLKKTSDGWATTLNNKELKAEGQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CasX-gRNA-DNA(45bp) state I / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Deltaproteobacteria bacterium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 46 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149361 / Symmetry type: POINT

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