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- EMDB-63767: Cryo-EM structure of wild-type SaCas9-guide RNA-mismatched target... -

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Basic information

Entry
Database: EMDB / ID: EMD-63767
TitleCryo-EM structure of wild-type SaCas9-guide RNA-mismatched target DNA complex
Map data
Sample
  • Complex: Wild-type SaCas9-sgRNA-mismatched target DNA ternary complex
    • DNA: target DNA strand
    • Protein or peptide: Ubiquitin-like protein SMT3,CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • DNA: non-target DNA strand
KeywordsCRISPR-CAS9 / GENOME ENGINEERING / HYDROLASE-RNA-DNA COMPLEX / DNA BINDING PROTEIN
Function / homology
Function and homology information


SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / SUMO is proteolytically processed / SUMOylation of transcription factors / Postmitotic nuclear pore complex (NPC) reformation / SUMOylation of transcription cofactors / septin ring / SUMOylation of DNA damage response and repair proteins / Transcriptional and post-translational regulation of MITF-M expression and activity ...SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / SUMO is proteolytically processed / SUMOylation of transcription factors / Postmitotic nuclear pore complex (NPC) reformation / SUMOylation of transcription cofactors / septin ring / SUMOylation of DNA damage response and repair proteins / Transcriptional and post-translational regulation of MITF-M expression and activity / SUMOylation of DNA replication proteins / SUMOylation of SUMOylation proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / SUMOylation of RNA binding proteins / SUMOylation of chromatin organization proteins / maintenance of CRISPR repeat elements / ubiquitin-like protein ligase binding / protein sumoylation / condensed nuclear chromosome / protein tag activity / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding / identical protein binding / nucleus
Similarity search - Function
: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / Rad60/SUMO-like domain / Ubiquitin-2 like Rad60 SUMO-like / : / Cas9 RuvC domain ...: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / Rad60/SUMO-like domain / Ubiquitin-2 like Rad60 SUMO-like / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ribonuclease H superfamily / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9 / Small ubiquitin-related modifier
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsNakagawa R / Omura SN / Yamashita K / Nishimasu H / Nureki O
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: To Be Published
Title: Molecular engineering and structural mechanism of the compact CRISPR-Cas9 nuclease
Authors: Nakagawa R / Omura SN / Kajimoto S / Okazaki S / Ishiguro S / Mori H / Kashiwakura Y / Hiramoto T / Hirano H / Yamashita K / Jividen K / Tsai SQ / Yachie N / Ohmori T / Nishimasu H / Nureki O
History
DepositionMar 15, 2025-
Header (metadata) releaseMar 18, 2026-
Map releaseMar 18, 2026-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_63767.png

Downloads & links

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Map

FileDownload / File: emd_63767.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 150 pix.
= 160.065 Å		1.07 Å/pix.
x 150 pix.
= 160.065 Å		1.07 Å/pix.
x 150 pix.
= 160.065 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0671 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.36585778 - 0.81186336
Average (Standard dev.)0.0029099276 (±0.026070405)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin656565
Dimensions150150150
Spacing150150150
CellA=B=C: 160.065 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_63767_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_63767_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_63767_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Wild-type SaCas9-sgRNA-mismatched target DNA ternary complex

EntireName: Wild-type SaCas9-sgRNA-mismatched target DNA ternary complex
Components
  • Complex: Wild-type SaCas9-sgRNA-mismatched target DNA ternary complex
    • DNA: target DNA strand
    • Protein or peptide: Ubiquitin-like protein SMT3,CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • DNA: non-target DNA strand

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Supramolecule #1: Wild-type SaCas9-sgRNA-mismatched target DNA ternary complex

SupramoleculeName: Wild-type SaCas9-sgRNA-mismatched target DNA ternary complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Staphylococcus aureus (bacteria)

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Macromolecule #1: target DNA strand

MacromoleculeName: target DNA strand / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 13.304967 KDa
SequenceString:
(DA)(DT)(DT)(DT)(DG)(DA)(DA)(DT)(DA)(DT) (DT)(DC)(DA)(DA)(DG)(SC)(SC)(AS)(AS)(GS) (SC)(GS)(DC)(DA)(DC)(DC)(DT)(DA)(DA) (DT)(DT)(DT)(DC)(DC)(DG)(DG)(DT)(DA)(DT) (DT) (DT)(DA)(DG)

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Macromolecule #4: non-target DNA strand

MacromoleculeName: non-target DNA strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 13.434055 KDa
SequenceString:
(DC)(DT)(DA)(DA)(DA)(DT)(DA)(DC)(DG)(DG) (DG)(DA)(DA)(DA)(DT)(DT)(DA)(DG)(DG)(DT) (DG)(SC)(GS)(SC)(PST)(PST)(GS)(GS)(DC) (DT)(DT)(DG)(DA)(DA)(DT)(DA)(DT)(DT)(DC) (DA)(DA)(DA)(DT)

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Macromolecule #2: Ubiquitin-like protein SMT3,CRISPR-associated endonuclease Cas9

MacromoleculeName: Ubiquitin-like protein SMT3,CRISPR-associated endonuclease Cas9
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 137.650594 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHGS LQDSEVNQEA KPEVKPEVKP ETHINLKVSD GSSEIFFKIK KTTPLRRLME AFAKRQGKEM DSLRFLYDGI RIQADQAPE DLDMEDNDII EAHREQIGGE NLYFQGSHMK RNYILGLDIG ITSVGYGIID YETRDVIDAG VRLFKEANVE N NEGRRSKR ...String:
MGHHHHHHGS LQDSEVNQEA KPEVKPEVKP ETHINLKVSD GSSEIFFKIK KTTPLRRLME AFAKRQGKEM DSLRFLYDGI RIQADQAPE DLDMEDNDII EAHREQIGGE NLYFQGSHMK RNYILGLDIG ITSVGYGIID YETRDVIDAG VRLFKEANVE N NEGRRSKR GARRLKRRRR HRIQRVKKLL FDYNLLTDHS ELSGINPYEA RVKGLSQKLS EEEFSAALLH LAKRRGVHNV NE VEEDTGN ELSTKEQISR NSKALEEKYV AELQLERLKK DGEVRGSINR FKTSDYVKEA KQLLKVQKAY HQLDQSFIDT YID LLETRR TYYEGPGEGS PFGWKDIKEW YEMLMGHCTY FPEELRSVKY AYNADLYNAL NDLNNLVITR DENEKLEYYE KFQI IENVF KQKKKPTLKQ IAKEILVNEE DIKGYRVTST GKPEFTNLKV YHDIKDITAR KEIIENAELL DQIAKILTIY QSSED IQEE LTNLNSELTQ EEIEQISNLK GYTGTHNLSL KAINLILDEL WHTNDNQIAI FNRLKLVPKK VDLSQQKEIP TTLVDD FIL SPVVKRSFIQ SIKVINAIIK KYGLPNDIII ELAREKNSKD AQKMINEMQK RNRQTNERIE EIIRTTGKEN AKYLIEK IK LHDMQEGKCL YSLEAIPLED LLNNPFNYEV DHIIPRSVSF DNSFNNKVLV KQEENSKKGN RTPFQYLSSS DSKISYET F KKHILNLAKG KGRISKTKKE YLLEERDINR FSVQKDFINR NLVDTRYATR GLMNLLRSYF RVNNLDVKVK SINGGFTSF LRRKWKFKKE RNKGYKHHAE DALIIANADF IFKEWKKLDK AKKVMENQMF EEKQAESMPE IETEQEYKEI FITPHQIKHI KDFKDYKYS HRVDKKPNRE LINDTLYSTR KDDKGNTLIV NNLNGLYDKD NDKLKKLINK SPEKLLMYHH DPQTYQKLKL I MEQYGDEK NPLYKYYEET GNYLTKYSKK DNGPVIKKIK YYGNKLNAHL DITDDYPNSR NKVVKLSLKP YRFDVYLDNG VY KFVTVKN LDVIKKENYY EVNSKCYEEA KKLKKISNQA EFIASFYNND LIKINGELYR VIGVNNDLLN RIEVNMIDIT YRE YLENMN DKRPPRIIKT IASKTQSIKK YSTDILGNLY EVKSKKHPQI IKKG

UniProtKB: Small ubiquitin-related modifier, CRISPR-associated endonuclease Cas9

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Macromolecule #3: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 31.892889 KDa
SequenceString:
GGGAAAUUAG GUGCGCUUGG CGUUUUAGUA CUCUGGAAAC AGAAUCUACU AAAACAAGGC AAAAUGCCGU GUUUAUCUCG UCAACUUGU UGGCGAGAUA

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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

BufferpH: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 101789
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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