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- EMDB-17702: Cas9 bound to cognate DNA, Streptococcus thermophilus DGCC 7710 C... -

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Basic information

Entry
Database: EMDB / ID: EMD-17702
TitleCas9 bound to cognate DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system
Map dataMap sharpened using phenix.auto_sharpen_1.21rc1-4903 using _iso_to_d_cut procedure and 3.24A cutoff.
Sample
  • Complex: Complex of Cas9 bound to crRNA:tracrRNA and to a cognate DNA duplex
    • Complex: CRISPR-associated endonuclease Cas9
      • Protein or peptide: CRISPR-associated endonuclease Cas9
    • Complex: crRNA, chain B and tracrRNA, chain C
      • RNA: crRNA, chain B
      • RNA: tracrRNA, chain C
    • Complex: DNA oligoduplex, target strand, chains D,E and DNA oligoduplex, non-target strand, chain F
      • DNA: DNA oligoduplex, target strand, chains D,E
      • DNA: DNA oligoduplex, non-target strand, chain F
  • Ligand: MAGNESIUM ION
KeywordsCas9 / CRISPR-Cas / PAM / spacer acquisition / RNA BINDING PROTEIN
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / hydrolase activity / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. ...CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / 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 speciesStreptococcus thermophilus DGCC 7710 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsSasnauskas G / Gaizauskaite U / Tamulaitiene G
Funding supportLithuania, 1 items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-19-32Lithuania
CitationJournal: Mol Cell / Year: 2026
Title: Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation.
Authors: Ugne Gaizauskaite / Giedre Tamulaitiene / Arunas Silanskas / Giedrius Gasiunas / Virginijus Siksnys / Giedrius Sasnauskas
Abstract: During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer ...During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems, selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a "supercomplex" with the Cas1-Cas2 integrase and the Csn2 protein. Here, we present cryo-electron microscopy structures of the Streptococcus thermophilus type II-A prespacer selection supercomplex in the DNA-scanning and two distinct PAM-bound configurations, providing insights into the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems. Repurposing Cas9 by the CRISPR adaptation machinery for prespacer selection, as characterized here, demonstrates Cas9 plasticity and expands our knowledge of Cas9 biology.
History
DepositionJun 23, 2023-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateMar 4, 2026-
Current statusMar 4, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17702.png

Downloads & links

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Map

FileDownload / File: emd_17702.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap sharpened using phenix.auto_sharpen_1.21rc1-4903 using _iso_to_d_cut procedure and 3.24A cutoff.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.1 Å/pix.
x 200 pix.
= 220. Å		1.1 Å/pix.
x 200 pix.
= 220. Å		1.1 Å/pix.
x 200 pix.
= 220. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-22.785442 - 35.520446999999997
Average (Standard dev.)0.00000000000844 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 220.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17702_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened map

Fileemd_17702_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17702_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_17702_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : Complex of Cas9 bound to crRNA:tracrRNA and to a cognate DNA duplex

EntireName: Complex of Cas9 bound to crRNA:tracrRNA and to a cognate DNA duplex
Components
  • Complex: Complex of Cas9 bound to crRNA:tracrRNA and to a cognate DNA duplex
    • Complex: CRISPR-associated endonuclease Cas9
      • Protein or peptide: CRISPR-associated endonuclease Cas9
    • Complex: crRNA, chain B and tracrRNA, chain C
      • RNA: crRNA, chain B
      • RNA: tracrRNA, chain C
    • Complex: DNA oligoduplex, target strand, chains D,E and DNA oligoduplex, non-target strand, chain F
      • DNA: DNA oligoduplex, target strand, chains D,E
      • DNA: DNA oligoduplex, non-target strand, chain F
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of Cas9 bound to crRNA:tracrRNA and to a cognate DNA duplex

SupramoleculeName: Complex of Cas9 bound to crRNA:tracrRNA and to a cognate DNA duplex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5

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Supramolecule #2: CRISPR-associated endonuclease Cas9

SupramoleculeName: CRISPR-associated endonuclease Cas9 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)

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Supramolecule #3: crRNA, chain B and tracrRNA, chain C

SupramoleculeName: crRNA, chain B and tracrRNA, chain C / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)

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Supramolecule #4: DNA oligoduplex, target strand, chains D,E and DNA oligoduplex, n...

SupramoleculeName: DNA oligoduplex, target strand, chains D,E and DNA oligoduplex, non-target strand, chain F
type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4-#5
Source (natural)Organism: synthetic construct (others)

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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: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 162.498531 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. DH10B (bacteria)
SequenceString: MTKPYSIGLD IGTNSVGWAV ITDNYKVPSK KMKVLGNTSK KYIKKNLLGV LLFDSGITAE GRRLKRTARR RYTRRRNRIL YLQEIFSTE MATLDDAFFQ RLDDSFLVPD DKRDSKYPIF GNLVEEKVYH DEFPTIYHLR KYLADSTKKA DLRLVYLALA H MIKYRGHF ...String:
MTKPYSIGLD IGTNSVGWAV ITDNYKVPSK KMKVLGNTSK KYIKKNLLGV LLFDSGITAE GRRLKRTARR RYTRRRNRIL YLQEIFSTE MATLDDAFFQ RLDDSFLVPD DKRDSKYPIF GNLVEEKVYH DEFPTIYHLR KYLADSTKKA DLRLVYLALA H MIKYRGHF LIEGEFNSKN NDIQKNFQDF LDTYNAIFES DLSLENSKQL EEIVKDKISK LEKKDRILKL FPGEKNSGIF SE FLKLIVG NQADFRKCFN LDEKASLHFS KESYDEDLET LLGYIGDDYS DVFLKAKKLY DAILLSGFLT VTDNETEAPL SSA MIKRYN EHKEDLALLK EYIRNISLKT YNEVFKDDTK NGYAGYIDGK TNQEDFYVYL KNLLAEFEGA DYFLEKIDRE DFLR KQRTF DNGSIPYQIH LQEMRAILDK QAKFYPFLAK NKERIEKILT FRIPYYVGPL ARGNSDFAWS IRKRNEKITP WNFED VIDK ESSAEAFINR MTSFDLYLPE EKVLPKHSLL YETFNVYNEL TKVRFIAESM RDYQFLDSKQ KKDIVRLYFK DKRKVT DKD IIEYLHAIYG YDGIELKGIE KQFNSSLSTY HDLLNIINDK EFLDDSSNEA IIEEIIHTLT IFEDREMIKQ RLSKFEN IF DKSVLKKLSR RHYTGWGKLS AKLINGIRDE KSGNTILDYL IDDGISNRNF MQLIHDDALS FKKKIQKAQI IGDEDKGN I KEVVKSLPGS PAIKKGILQS IKIVDELVKV MGGRKPESIV VEMARENQYT NQGKSNSQQR LKRLEKSLKE LGSKILKEN IPAKLSKIDN NALQNDRLYL YYLQNGKDMY TGDDLDIDRL SNYDIDHIIP QAFLKDNSID NKVLVSSASN RGKSDDFPSL EVVKKRKTF WYQLLKSKLI SQRKFDNLTK AERGGLLPED KAGFIQRQLV ETRQITKHVA RLLDEKFNNK KDENNRAVRT V KIITLKST LVSQFRKDFE LYKVREINDF HHAHDAYLNA VIASALLKKY PKLEPEFVYG DYPKYNSFRE RKSATEKVYF YS NIMNIFK KSISLADGRV IERPLIEVNE ETGESVWNKE SDLATVRRVL SYPQVNVVKK VEEQNHGLDR GKPKGLFNAN LSS KPKPNS NENLVGAKEY LDPKKYGGYA GISNSFAVLV KGTIEKGAKK KITNVLEFQG ISILDRINYR KDKLNFLLEK GYKD IELII ELPKYSLFEL SDGSRRMLAS ILSTNNKRGE IHKGNQIFLS QKFVKLLYHA KRISNTINEN HRKYVENHKK EFEEL FYYI LEFNENYVGA KKNGKLLNSA FQSWQNHSID ELCSSFIGPT GSERKGLFEL TSRGSAADFE FLGVKIPRYR DYTPSS LLK DATLIHQSVT GLYETRIDLA KLGEGLEGHH HHHH

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #2: crRNA, chain B

MacromoleculeName: crRNA, chain B / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 13.609083 KDa
SequenceString:
CGCUAAAGAG GAAGAGGACA GUUUUAGAGC UGUGUUGUUU CG

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Macromolecule #3: tracrRNA, chain C

MacromoleculeName: tracrRNA, chain C / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 24.119357 KDa
SequenceString:
CGAAACAACA CAGCGAGUUA AAAUAAGGCU UAGUCCGUAC UCAACUUGAA AAGGUGGCAC CGAUUCGGUG UUUUU

GENBANK: GENBANK: HQ712120.1

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Macromolecule #4: DNA oligoduplex, target strand, chains D,E

MacromoleculeName: DNA oligoduplex, target strand, chains D,E / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.670199 KDa
SequenceString:
(DG)(DC)(DT)(DG)(DA)(DC)(DT)(DC)(DA)(DC) (DC)(DA)(DT)(DG)(DT)(DC)(DC)(DT)(DC)(DT) (DT)(DC)(DC)(DT)(DC)(DT)(DT)(DT)(DA) (DG)(DC)(DG)

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Macromolecule #5: DNA oligoduplex, non-target strand, chain F

MacromoleculeName: DNA oligoduplex, non-target strand, chain F / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.718427 KDa
SequenceString:
(DT)(DG)(DG)(DT)(DG)(DA)(DG)(DT)(DC)(DA) (DG)(DC)

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1744 / Average exposure time: 46.33 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 92000
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 894269 / Details: Blob picking in cryoSPARC live session
CTF correctionSoftware - Name: cryoSPARC (ver. 4.0.3) / Type: NONE
Startup modelType of model: INSILICO MODEL / In silico model: ab-initio cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.1) / Number images used: 119790
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-8pj9:
Cas9 bound to cognate DNA, Streptococcus thermophilus DGCC 7710 CRISPR3 system

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