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- EMDB-52320: PAM-bound Cas9-Cas1-Cas2-Csn2 supercomplex in the docked conforma... -

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Basic information

Entry
Database: EMDB / ID: EMD-52320
TitlePAM-bound Cas9-Cas1-Cas2-Csn2 supercomplex in the docked conformation, Streptococcus thermophilus DGCC 7710 CRISPR3 system
Map dataUnsharpened composite map
Sample
  • Complex: PAM-bound Cas9-Cas1-Cas2-Csn2 supercomplex in the docked conformation
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: Cas9 crRNA
    • RNA: Cas9 tracrRNA
    • DNA: DNA (43-MER)
    • DNA: DNA (43-MER)
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • Protein or peptide: CRISPR-associated protein Csn2
  • Ligand: CALCIUM ION
KeywordsCIRSPR-Cas / spacer acquisition / Csn2 / Cas1 / Cas2 / CRISPR integrase / DNA BINDING PROTEIN
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / RNA endonuclease activity / DNA endonuclease activity / 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 protein, Csn2-type / CRISPR-associated protein Csn2 superfamily / CRISPR-associated protein (Cas_Csn2) / CRISPR-associated protein Cas1, NMENI subtype / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain ...CRISPR-associated protein, Csn2-type / CRISPR-associated protein Csn2 superfamily / CRISPR-associated protein (Cas_Csn2) / CRISPR-associated protein Cas1, NMENI subtype / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1 / 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 / CRISPR-associated endonuclease Cas1 / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated protein Csn2
Similarity search - Component
Biological speciesStreptococcus thermophilus DGCC 7710 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 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
DepositionDec 12, 2024-
Header (metadata) releaseFeb 18, 2026-
Map releaseFeb 18, 2026-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52320.png

Downloads & links

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Map

FileDownload / File: emd_52320.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened composite map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.1 Å/pix.
x 336 pix.
= 369.6 Å		1.1 Å/pix.
x 336 pix.
= 369.6 Å		1.1 Å/pix.
x 336 pix.
= 369.6 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: 6.31
Minimum - Maximum-7.4482207 - 30.654668999999998
Average (Standard dev.)0.0012154306 (±1.0103464)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 369.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened composite map (phenix.auto sharpen 1.21.2-5419, b iso to d cut, 3.3A)...

Fileemd_52320_additional_1.map
AnnotationSharpened composite map (phenix.auto_sharpen_1.21.2-5419, b_iso_to_d_cut, 3.3A)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PAM-bound Cas9-Cas1-Cas2-Csn2 supercomplex in the docked conformation

EntireName: PAM-bound Cas9-Cas1-Cas2-Csn2 supercomplex in the docked conformation
Components
  • Complex: PAM-bound Cas9-Cas1-Cas2-Csn2 supercomplex in the docked conformation
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: Cas9 crRNA
    • RNA: Cas9 tracrRNA
    • DNA: DNA (43-MER)
    • DNA: DNA (43-MER)
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: CRISPR-associated endoribonuclease Cas2
    • Protein or peptide: CRISPR-associated protein Csn2
  • Ligand: CALCIUM ION

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Supramolecule #1: PAM-bound Cas9-Cas1-Cas2-Csn2 supercomplex in the docked conformation

SupramoleculeName: PAM-bound Cas9-Cas1-Cas2-Csn2 supercomplex in the docked conformation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Details: C-terminal His-Tag / 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 BL21(DE3) (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 #6: CRISPR-associated endonuclease Cas1

MacromoleculeName: CRISPR-associated endonuclease Cas1 / type: protein_or_peptide / ID: 6 / Details: C-terminal His-tag / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 34.854875 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAGWRTVVVN IHSKLSYKNN HLIFRNSYKT EMIHLSEIDI LLLETTDIVL TTMLVKRLVD ENILVIFCDD KRLPTAFLTP YYARHDSSL QIARQIAWKE NVKCEVWTAI IAQKILNQSY YLGECSFFEK SQSIMELYHG LERFDPSNRE GHSARIYFNT L FGNDFTRE ...String:
MAGWRTVVVN IHSKLSYKNN HLIFRNSYKT EMIHLSEIDI LLLETTDIVL TTMLVKRLVD ENILVIFCDD KRLPTAFLTP YYARHDSSL QIARQIAWKE NVKCEVWTAI IAQKILNQSY YLGECSFFEK SQSIMELYHG LERFDPSNRE GHSARIYFNT L FGNDFTRE SDNDINAALD YGYTLLLSMF AREVVVCGCM TQIGLKHANQ FNQFNLASDI MEPFRPIIDR IVYQNRHNNF VK IKKELFS IFSETYLYNG KEMYLSNIVS DYTKKVIKAL NQLGEEIPEF RMSGHHHHHH

UniProtKB: CRISPR-associated endonuclease Cas1

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Macromolecule #7: CRISPR-associated endoribonuclease Cas2

MacromoleculeName: CRISPR-associated endoribonuclease Cas2 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 13.43156 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSYRYMRMIL MFDMPTDTAE ERKAYRKFRK FLLSEGFIMH QFSVYSKLLL NHTANTAMVG RLKANNPKKG NITILTVTEK QFARMIYLY GDKNTSIANS EERLVFLGDN YCDED

UniProtKB: CRISPR-associated endoribonuclease Cas2

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Macromolecule #8: CRISPR-associated protein Csn2

MacromoleculeName: CRISPR-associated protein Csn2 / type: protein_or_peptide / ID: 8 / Details: N-terminal His-tag / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 27.459561 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGHHHHHHEN LYFQGGMKIN FSLLDEPMEV NLGTVLVIED VSVFAQLVKE FYQYDEQSNL TIFDSKIRSI RSSELLLITD ILGYDINTS QVLKLLHTDI VSQLNDKPEV RSEIDSLVSL ITDIIMAECI ENELDIEYDE ITLLELIKAL GVRIETKSCT V FEKIFEIL ...String:
MGHHHHHHEN LYFQGGMKIN FSLLDEPMEV NLGTVLVIED VSVFAQLVKE FYQYDEQSNL TIFDSKIRSI RSSELLLITD ILGYDINTS QVLKLLHTDI VSQLNDKPEV RSEIDSLVSL ITDIIMAECI ENELDIEYDE ITLLELIKAL GVRIETKSCT V FEKIFEIL QIFKYLVKKR ILVFVNSLSY FSKDEIYQIL EYTKLSQADV LFLEPRQIEG IQQFILDKDY ILMPYNN

UniProtKB: CRISPR-associated protein Csn2

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Macromolecule #2: Cas9 crRNA

MacromoleculeName: Cas9 crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.609083 KDa
SequenceString:
CGCUAAAGAG GAAGAGGACA GUUUUAGAGC UGUGUUGUUU CG

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Macromolecule #3: Cas9 tracrRNA

MacromoleculeName: Cas9 tracrRNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 24.119357 KDa
SequenceString:
CGAAACAACA CAGCGAGUUA AAAUAAGGCU UAGUCCGUAC UCAACUUGAA AAGGUGGCAC CGAUUCGGUG UUUUU

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Macromolecule #4: DNA (43-MER)

MacromoleculeName: DNA (43-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.003586 KDa
SequenceString:
(DG)(DA)(DT)(DG)(DT)(DC)(DT)(DT)(DC)(DC) (DA)(DA)(DT)(DT)(DT)(DA)(DC)(DT)(DA)(DC) (DT)(DC)(DG)(DT)(DT)(DC)(DT)(DG)(DG) (DT)(DG)(DT)(DT)(DT)(DC)(DT)(DC)(DG)(DA) (DG) (DG)(DC)(DG)(DT)(DC)(DA)(DG)(DC) (DC)

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Macromolecule #5: DNA (43-MER)

MacromoleculeName: DNA (43-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.182787 KDa
SequenceString:
(DG)(DG)(DC)(DT)(DG)(DA)(DC)(DG)(DC)(DC) (DT)(DC)(DG)(DA)(DG)(DA)(DA)(DA)(DC)(DA) (DC)(DC)(DA)(DG)(DA)(DA)(DC)(DG)(DA) (DG)(DT)(DA)(DG)(DT)(DA)(DA)(DA)(DT)(DT) (DG) (DG)(DA)(DA)(DG)(DA)(DC)(DA)(DT) (DC)

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Macromolecule #9: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 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. / Pretreatment - Atmosphere: AIR / Details: Glowcube plus, 20 mA
VitrificationCryogen name: ETHANE / 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 / Average exposure time: 46.33 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: cryoSPARC ab-initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 26925
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

9h2g


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-9hp8:
PAM-bound Cas9-Cas1-Cas2-Csn2 supercomplex in the docked conformation, Streptococcus thermophilus DGCC 7710 CRISPR3 system

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