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- PDB-7way: PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state -

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Basic information

Entry
Database: PDB / ID: 7way
TitlePlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state
Components
  • DNA (27-MER)
  • DNA (33-MER)
  • RNA
  • dPlmCasX
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR / CasX / sgRNA / R-loop complex / RNA BINDING PROTEIN-DNA-RNA complex / RNA BINDING PROTEIN / DNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Transposase
Function and homology information
Biological speciesPlanctomycetes bacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsZhang, S. / Liu, J.J.G.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Mol Cell / Year: 2022
Title: Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.
Authors: Connor A Tsuchida / Shouyue Zhang / Mohammad Saffari Doost / Yuqian Zhao / Jia Wang / Elizabeth O'Brien / Huan Fang / Cheng-Ping Li / Danyuan Li / Zhuo-Yan Hai / Jonathan Chuck / Julian ...Authors: Connor A Tsuchida / Shouyue Zhang / Mohammad Saffari Doost / Yuqian Zhao / Jia Wang / Elizabeth O'Brien / Huan Fang / Cheng-Ping Li / Danyuan Li / Zhuo-Yan Hai / Jonathan Chuck / Julian Brötzmann / Araz Vartoumian / David Burstein / Xiao-Wei Chen / Eva Nogales / Jennifer A Doudna / Jun-Jie Gogo Liu /
Abstract: A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.
History
DepositionDec 15, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 16, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 30, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jun 26, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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  • EMDB-32389
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Assembly

Deposited unit
B: DNA (33-MER)
C: DNA (27-MER)
D: RNA
A: dPlmCasX


Theoretical massNumber of molelcules
Total (without water)176,4334
Polymers176,4334
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21210 Å2
ΔGint-101 kcal/mol
Surface area71270 Å2

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Components

#1: DNA chain DNA (33-MER)


Mass: 12283.895 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Planctomycetes bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#2: DNA chain DNA (27-MER)


Mass: 12193.829 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Planctomycetes bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#3: RNA chain RNA


Mass: 39272.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Planctomycetes bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#4: Protein dPlmCasX


Mass: 112682.570 Da / Num. of mol.: 1 / Mutation: D659A, E756A, E922A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Planctomycetes bacterium (bacteria) / Gene: A3G70_06685 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1G3BXR9

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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

Component
IDNameTypeEntity IDParent-IDSource
1PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading stateCOMPLEXall0RECOMBINANT
2dsDNACOMPLEX#1-#21RECOMBINANT
3sgRNAv1COMPLEX#31RECOMBINANT
4PlmCasXCOMPLEX#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Planctomycetes (bacteria)203682
32Planctomycetes (bacteria)203682
43Planctomycetes (bacteria)203682
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: cryoSPARC / Version: 2 / Category: final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 520000 / Symmetry type: POINT
Atomic model buildingB value: 57 / Protocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412051
ELECTRON MICROSCOPYf_angle_d0.52917051
ELECTRON MICROSCOPYf_dihedral_angle_d17.7623023
ELECTRON MICROSCOPYf_chiral_restr0.0361961
ELECTRON MICROSCOPYf_plane_restr0.0041546

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