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- EMDB-39942: Cryo-EM structure of eSaCas9_NNG-guide RNA-target DNA complex in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-39942
TitleCryo-EM structure of eSaCas9_NNG-guide RNA-target DNA complex in an interrogation state
Map dataLocally filtered a sharpened map using a local resolution map.
Sample
  • Complex: eSaCas9-NNG-sgRNA-target DNA ternary complex in an intermediaitie state
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
KeywordsCRISPR-CAS9 / GENOME ENGINEERING / HYDROLASE-RNA-DNA COMPLEX / DNA BINDING PROTEIN
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsOmura SN / Nakagawa R / 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: Omura SN / Nakagawa R / 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
DepositionApr 30, 2024-
Header (metadata) releaseNov 5, 2025-
Map releaseNov 5, 2025-
UpdateNov 5, 2025-
Current statusNov 5, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39942.png

Downloads & links

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Map

FileDownload / File: emd_39942.map.gz / Format: CCP4 / Size: 13.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally filtered a sharpened map using a local resolution map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 154 pix.
= 170.432 Å		1.11 Å/pix.
x 154 pix.
= 170.432 Å		1.11 Å/pix.
x 154 pix.
= 170.432 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1067 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.46317148 - 0.7884243
Average (Standard dev.)0.0025240323 (±0.022741605)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin585858
Dimensions154154154
Spacing154154154
CellA=B=C: 170.4318 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_39942_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_39942_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_39942_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 : eSaCas9-NNG-sgRNA-target DNA ternary complex in an intermediaitie...

EntireName: eSaCas9-NNG-sgRNA-target DNA ternary complex in an intermediaitie state
Components
  • Complex: eSaCas9-NNG-sgRNA-target DNA ternary complex in an intermediaitie state
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand

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Supramolecule #1: eSaCas9-NNG-sgRNA-target DNA ternary complex in an intermediaitie...

SupramoleculeName: eSaCas9-NNG-sgRNA-target DNA ternary complex in an intermediaitie state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 124.305898 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSHMKRNYIL GLDIGITSVG YGIIDYETRD VIDAGVRLFK EANVENNEGR RSKRGARRLK RRRRHRIQRV KKLLFDYNLL TDHSELSGI NPYEARVKGL SQKLSEEEFS AALLHLAKRR GVHNVNEVEE DTGNELSTKE QISRNSKALE EKYVAELQLE R LKKDGEVR ...String:
GSHMKRNYIL GLDIGITSVG YGIIDYETRD VIDAGVRLFK EANVENNEGR RSKRGARRLK RRRRHRIQRV KKLLFDYNLL TDHSELSGI NPYEARVKGL SQKLSEEEFS AALLHLAKRR GVHNVNEVEE DTGNELSTKE QISRNSKALE EKYVAELQLE R LKKDGEVR GSINRFKTSD YVKEAKQLLK VQKAYHQLDQ SFIDTYIDLL ETRRTYYEGP GEGSPFGWKD IKEWYEMLMG HC TYFPEEL RSVKYAYNAD LYNALNDLNN LVITRDENEK LEYYEKFQII ENVFKQKKKP TLKQIAKEIL VNEEDIKGYR VTS TGKPEF TNLKVYHDIK DITARKEIIE NAELLDQIAK ILTIYQSSED IQEELTNLNS ELTQEEIEQI SNLKGYTGTH NLSL KAINL ILDELWHTND AQIAIFNALK LVPKKVDLSQ QKEIPTTLVD DFILSPVVKR SFIQSIKVIN AIIKKYGLPN DIIIE LARE KNSKDAQKMI NEMQKRNRQT NERIEEIIRT TGKENAKYLI EKIKLHDMQE GKCLYSLEAI PLEDLLNNPF NYEVDH IIP RSVSFDNSFN NKVLVKQEEN SKKGNRTPFQ YLSSSDSKIS YETFKKHILN LAKGKGRISK TKKEYLLEER DINRFSV QK DFINRNLVDT RYATRGLMNL LRSYFRVNNL DVKVKSINGG FTSFLRRKWK FKKERNKGYK HHAEDALIIA NADFIFKE W KKLDKAKKVM ENQMFEEKQA ESMPEIETEQ EYKEIFITPH QIKHIKDFKD YKYSHRVDKK PNRKLINDTL YSTRKDDKG NTRIVNNLNG LYDKDNDKLK KLINKSPEKL LMYHHDPQTY QKLKLIMEQY GDEKNPLYKY YEETGNYLTK YSKKDNGPVI KKIKYYGNK LNAHLDITDD YPNSRNKVVK LSLKPYRFDV YLDNGVYKFV KVNNLDVIKK ENYYEVNSKC YEEAKKLKKI S NQAEFIAS FYRNDLIKIN GELYRVIGVA SDLLNAIEVN MIDITYREYL ENMNDKRPPR IFKTISSKTQ SIKKYSTDIL GN LYEVKSK KHPQIIKKG

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / 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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Macromolecule #3: Target DNA strand

MacromoleculeName: Target DNA strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 13.314969 KDa
SequenceString:
(DA)(DT)(DT)(DT)(DG)(DA)(DA)(DT)(DA)(DG) (DG)(DC)(DA)(DA)(DG)(SC)(SC)(AS)(AS)(GS) (SC)(GS)(DC)(DA)(DC)(DC)(DT)(DA)(DA) (DT)(DT)(DT)(DC)(DC)(DC)(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: 13.386006 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)(DC)(DC)(DT)(DA)(DT)(DT)(DC) (DA)(DA)(DA)(DT)

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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: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 156154
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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