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- PDB-8ywh: Cryo-EM structure of small and dead form SaCas9-RNA-DNA ternary c... -

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Basic information

Entry
Database: PDB / ID: 8ywh
TitleCryo-EM structure of small and dead form SaCas9-RNA-DNA ternary complex (sdCas9)
Components
  • Non-target DNA
  • Target DNA
  • sCas9 (Compact SaCas9)
  • sgRNA
KeywordsDNA BINDING PROTEIN / CRISPR/Cas9 / Thermostable protein engineering / Domain minimized Cas / engineered SaCas9
Function / homology: / DNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesStaphylococcus aureus (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsKang, E.S. / Kim, N.H. / Thach, T.T. / Hyun, J. / Kim, Y.H.
Funding support Korea, Republic Of, 2items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)NRF-2023R1A2C300573 Korea, Republic Of
National Research Foundation (NRF, Korea)RS-2023-00302458 Korea, Republic Of
CitationJournal: Adv Mater / Year: 2025
Title: Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression.
Authors: Eun Sung Kang / Nam Hyeong Kim / Hyun-Kyoung Lim / Hyeyeon Jeon / Kayoung Han / Young Hyun No / Kyungtae Kim / Zinah Hilal Khaleel / Dongsun Shin / Kilho Eom / Jiyoung Nam / Bok-Soo Lee / ...Authors: Eun Sung Kang / Nam Hyeong Kim / Hyun-Kyoung Lim / Hyeyeon Jeon / Kayoung Han / Young Hyun No / Kyungtae Kim / Zinah Hilal Khaleel / Dongsun Shin / Kilho Eom / Jiyoung Nam / Bok-Soo Lee / Han-Joo Kim / Minah Suh / Jaecheol Lee / Trung Thanh Thach / Jaekyung Hyun / Yong Ho Kim /
Abstract: Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure- ...Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.
History
DepositionMar 31, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 25, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update
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 / Item: _em_admin.last_update
Revision 1.2Jul 16, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: sCas9 (Compact SaCas9)
B: sgRNA
C: Target DNA
D: Non-target DNA


Theoretical massNumber of molelcules
Total (without water)172,0474
Polymers172,0474
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein sCas9 (Compact SaCas9)


Mass: 104196.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Production host: Escherichia coli (E. coli)
#2: RNA chain sgRNA


Mass: 31483.635 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Target DNA


Mass: 18083.580 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: GenBank: 887958
#4: DNA chain Non-target DNA


Mass: 18283.701 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: GenBank: 887958
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

ComponentName: Cryo-EM structure of Compact SaCas9-RNA-DNA ternary complex
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.106 MDa / Experimental value: YES
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Details: 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 2 mM MgCl2, 1 mM TCEP
Buffer componentConc.: 20 mM / Name: Tris, NaCl, MgCl buffer / Formula: Tris-HCl
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Cas9: sgRNA: DNA ternary complex was generated by combining purified Cas9 protein, sgRNA, and dsDNA in molar ratios of 1:1.2:1.3, incubated at room temperature for 1 hour in 20 mM Tris-HCl, ...Details: Cas9: sgRNA: DNA ternary complex was generated by combining purified Cas9 protein, sgRNA, and dsDNA in molar ratios of 1:1.2:1.3, incubated at room temperature for 1 hour in 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 2 mM MgCl2, 1 mM TCEP.
Specimen supportDetails: -15 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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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: 2000 nm / Nominal defocus min: 700 nm / Cs: 0.1 mm / C2 aperture diameter: 70 µm
Image recordingAverage exposure time: 19.24 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 50
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.4.1particle selection
2cryoSPARC4.4.1image acquisition
3Topazimage acquisition
4RELION5image acquisition
5ResMap1.1.4image acquisition
7RELION5CTF correction
10PHENIX1.20.1model fitting4487-000
12RELION5initial Euler assignment
13RELION5final Euler assignment
14cryoSPARC4.4.1classification
15RELION53D reconstruction
16Coot0.8.9.3model refinement8011
23PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1758133
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162748 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingPDB-ID: 5AXW

5axw


Pdb chain-ID: a / Accession code: 5AXW / Chain residue range: 1-1053
Details: The initial model generated from 5AXW without HNH and Linker domain
Pdb chain residue range: 1-1053 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410429
ELECTRON MICROSCOPYf_angle_d0.6814703
ELECTRON MICROSCOPYf_dihedral_angle_d23.372811
ELECTRON MICROSCOPYf_chiral_restr0.0411706
ELECTRON MICROSCOPYf_plane_restr0.0041364

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