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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 / homologyUncharacterized protein
Function and homology information
Specimen sourceDeltaproteobacteria bacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.2 Å resolution
AuthorsLiu, J.J. / Orlova, N. / Nogales, E. / Doudna, J.A.
Funding supportUnited States , 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institutep01United 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 / Alexander Wagner / Julian Brötzmann / Brett T Staahl / Kian L Taylor / John Desmarais / Eva Nogales / Jennifer A Doudna
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 10, 2019 / Release: Feb 27, 2019

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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
Polyers174,9324
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)21450
ΔGint (kcal/M)-140
Surface area (Å2)66590

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Components

#1: DNA chain DNA target strand


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


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


Mass: 108092.836 Da / Num. of mol.: 1 / Mutation: D672A, E769A, D935A
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: (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 / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CasX-gRNA-DNA(45bp) state I / Type: COMPLEX / Entity ID: 1, 2, 3, 4 / 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
MicroscopyMicroscope model: 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 / Number of particles: 149361 / Symmetry type: POINT

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