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- PDB-7wb1: PlmCasX-sgRNAv2-dsDNA ternary complex at nts loading state -

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Basic information

Entry
Database: PDB / ID: 7wb1
TitlePlmCasX-sgRNAv2-dsDNA ternary complex at nts loading state
Components
  • NTS-DNA
  • RNA (121-MER)
  • TS-DNA
  • dPlmCasX
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR / CasX / sgRNA / R-loop 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: 3.7 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 1.0Mar 16, 2022Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 16, 2022Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 16, 2022Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Mar 16, 2022Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 16, 2022Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Mar 16, 2022Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 16, 2022Data content type: Primary map / Data content type: Primary map / Provider: 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
Revision 1.3Jun 25, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jun 25, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
B: TS-DNA
C: NTS-DNA
D: RNA (121-MER)
A: dPlmCasX


Theoretical massNumber of molelcules
Total (without water)176,0574
Polymers176,0574
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20320 Å2
ΔGint-95 kcal/mol
Surface area72860 Å2

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Components

#1: DNA chain TS-DNA


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 NTS-DNA


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 (121-MER)


Mass: 38897.156 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
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
Has protein modificationN

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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-sgRNAv2-dsDNA ternary complex at nts loading stateCOMPLEXall0RECOMBINANT
2dsDNACOMPLEX#1-#21RECOMBINANT
3sgRNAv2COMPLEX#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 software
IDNameVersionCategory
9PHENIXmodel refinement
11cryoSPARC2final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 616493 / Symmetry type: POINT
Atomic model buildingB value: 57 / Protocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312124
ELECTRON MICROSCOPYf_angle_d0.56817171
ELECTRON MICROSCOPYf_dihedral_angle_d21.4573068
ELECTRON MICROSCOPYf_chiral_restr0.0371979
ELECTRON MICROSCOPYf_plane_restr0.0041546

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