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- EMDB-39633: Cryo-EM structure of small and dead form SaCas9-RNA-DNA ternary c... -

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Basic information

Entry
Database: EMDB / ID: EMD-39633
TitleCryo-EM structure of small and dead form SaCas9-RNA-DNA ternary complex (sdCas9)
Map data
Sample
  • Complex: Cryo-EM structure of Compact SaCas9-RNA-DNA ternary complex
    • Protein or peptide: sCas9 (Compact SaCas9)
    • RNA: sgRNA
    • DNA: Target DNA
    • DNA: Non-target DNA
KeywordsCRISPR/Cas9 / Thermostable protein engineering / Domain minimized Cas / engineered SaCas9 / DNA BINDING PROTEIN
Biological speciesStaphylococcus aureus (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsKang ES / Kim NH / Thach TT / Hyun J / Kim YH
Funding support Korea, Republic Of, 2 items
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: 2024
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, 2024-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39633.png

Downloads & links

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Map

FileDownload / File: emd_39633.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.98 Å/pix.
x 256 pix.
= 249.6 Å		0.98 Å/pix.
x 256 pix.
= 249.6 Å		0.98 Å/pix.
x 256 pix.
= 249.6 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.975 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00164
Minimum - Maximum-0.035801042 - 0.061900724
Average (Standard dev.)0.00003269028 (±0.00092224835)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 249.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_39633_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_39633_half_map_2.map
Projections & Slices
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Sample components

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Entire : Cryo-EM structure of Compact SaCas9-RNA-DNA ternary complex

EntireName: Cryo-EM structure of Compact SaCas9-RNA-DNA ternary complex
Components
  • Complex: Cryo-EM structure of Compact SaCas9-RNA-DNA ternary complex
    • Protein or peptide: sCas9 (Compact SaCas9)
    • RNA: sgRNA
    • DNA: Target DNA
    • DNA: Non-target DNA

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Supramolecule #1: Cryo-EM structure of Compact SaCas9-RNA-DNA ternary complex

SupramoleculeName: Cryo-EM structure of Compact SaCas9-RNA-DNA ternary complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 106 KDa

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Macromolecule #1: sCas9 (Compact SaCas9)

MacromoleculeName: sCas9 (Compact SaCas9) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 104.196102 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKRNYILGLA IGITSVGYGI IDYETRDVID AGVRLFKEAN VENNEGRRSK RGARRLKRRR RHRIQRVKKL LFDYNLLTDH SELSGINPY EARVKGLSQK LSEEEFSAAL LHLAKRRGVH NVNEVEEDTG NELSTKEQIS RNSKALEEKY VAELQLERLK K DGEVRGSI ...String:
MKRNYILGLA IGITSVGYGI IDYETRDVID AGVRLFKEAN VENNEGRRSK RGARRLKRRR RHRIQRVKKL LFDYNLLTDH SELSGINPY EARVKGLSQK LSEEEFSAAL LHLAKRRGVH NVNEVEEDTG NELSTKEQIS RNSKALEEKY VAELQLERLK K DGEVRGSI NRFKTSDYVK EAKQLLKVQK AYHQLDQSFI DTYIDLLETR RTYYEGPGEG SPFGWKDIKE WYEMLMGHCT YF PEELRSV KYAYNADLYN ALNDLNNLVI TRDENEKLEY YEKFQIIENV FKQKKKPTLK QIAKEILVNE EDIKGYRVTS TGK PEFTNL KVYHDIKDIT ARKEIIENAE LLDQIAKILT IYQSSEDIQE ELTNLNSELT QEEIEQISNL KGYTGTHNLS LKAI NLILD ELWHTNDNQI AIFNRLKLVP KKVDLSQQKE IPTTLVDDFI LSPVVKRSFI QSIKVINAII KKYGLPNDII IELAR GGSY ATRGLMNLLR SYFRVNNLDV KVKSINGGFT SFLRRKWKFK KERNKGYKHH AEDALIIANA DFIFKEWKKL DKAKKV MEN QMFEEKQAES MPEIETEQEY KEIFITPHQI KHIKDFKDYK YSHRVDKKPN RELINDTLYS TRKDDKGNTL IVNNLNG LY DKDNDKLKKL INKSPEKLLM YHHDPQTYQK LKLIMEQYGD EKNPLYKYYE ETGNYLTKYS KKDNGPVIKK IKYYGNKL N AHLDITDDYP NSRNKVVKLS LKPYRFDVYL DNGVYKFVTV KNLDVIKKEN YYEVNSKCYE EAKKLKKISN QAEFIASFY NNDLIKINGE LYRVIGVNND LLNRIEVNMI DITYREYLEN MNDKRPPRII KTIASKTQSI KKYSTDILGN LYEVKSKKHP QIIKKG

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 31.483635 KDa
SequenceString:
GAUCUGAGUC CGGUAGCGCU AGUUUUAGUA CUCUGGAAAC AGAAUCUACU AAAACAAGGC AAAAUGCCGU GUUUAUCUCG UCAACUUGU UGGCGAGAU

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Macromolecule #3: Target DNA

MacromoleculeName: Target DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.08358 KDa
SequenceString: (DG)(DA)(DA)(DC)(DC)(DG)(DT)(DC)(DA)(DG) (DA)(DT)(DC)(DC)(DG)(DC)(DT)(DA)(DG)(DC) (DG)(DC)(DT)(DA)(DC)(DC)(DG)(DG)(DA) (DC)(DT)(DC)(DA)(DG)(DA)(DT)(DC)(DT)(DC) (DG) (DA)(DG)(DT)(DT)(DC)(DA) ...String:
(DG)(DA)(DA)(DC)(DC)(DG)(DT)(DC)(DA)(DG) (DA)(DT)(DC)(DC)(DG)(DC)(DT)(DA)(DG)(DC) (DG)(DC)(DT)(DA)(DC)(DC)(DG)(DG)(DA) (DC)(DT)(DC)(DA)(DG)(DA)(DT)(DC)(DT)(DC) (DG) (DA)(DG)(DT)(DT)(DC)(DA)(DA)(DG) (DC)(DT)(DT)(DC)(DG)(DA)(DA)(DT)(DT)(DC) (DT)

GENBANK: GENBANK: U19277.1

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Macromolecule #4: Non-target DNA

MacromoleculeName: Non-target DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.283701 KDa
SequenceString: (DA)(DG)(DA)(DA)(DT)(DT)(DC)(DG)(DA)(DA) (DG)(DC)(DT)(DT)(DG)(DA)(DA)(DC)(DT)(DC) (DG)(DA)(DG)(DA)(DT)(DC)(DT)(DG)(DA) (DG)(DT)(DC)(DC)(DG)(DG)(DT)(DA)(DG)(DC) (DG) (DC)(DT)(DA)(DG)(DC)(DG) ...String:
(DA)(DG)(DA)(DA)(DT)(DT)(DC)(DG)(DA)(DA) (DG)(DC)(DT)(DT)(DG)(DA)(DA)(DC)(DT)(DC) (DG)(DA)(DG)(DA)(DT)(DC)(DT)(DG)(DA) (DG)(DT)(DC)(DC)(DG)(DG)(DT)(DA)(DG)(DC) (DG) (DC)(DT)(DA)(DG)(DC)(DG)(DG)(DA) (DT)(DC)(DT)(DG)(DA)(DC)(DG)(DG)(DT)(DT) (DC)

GENBANK: GENBANK: U19277.1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 7.4 / Component - Concentration: 20.0 mM / Component - Formula: Tris-HCl / Component - Name: Tris, NaCl, MgCl buffer
Details: 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 2 mM MgCl2, 1 mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Details: -15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsCas9: 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 50 / Average exposure time: 19.24 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.1 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1758133
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5axw


Details: Crystal structure of Staphylococcus aureus Cas9 in complex with sgRNA and target DNA (TTGGGT PAM)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5.0) / Number images used: 162748
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5.0)
Final 3D classificationNumber classes: 50 / Software - Name: cryoSPARC (ver. 4.4.1)

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Atomic model buiding 1

Initial modelPDB ID:

5axw


Chain - Chain ID: a / Chain - Residue range: 1-1053 / Chain - Source name: PDB / Chain - Initial model type: experimental model
Details: The initial model generated from 5AXW without HNH and Linker domain
RefinementProtocol: AB INITIO MODEL
Output model

PDB-8ywh:
Cryo-EM structure of small and dead form SaCas9-RNA-DNA ternary complex (sdCas9)

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