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- EMDB-76399: Cryo-EM structure of dCas9 bound to DNA duplex with 10 bp extension -

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Basic information

Entry
Database: EMDB / ID: EMD-76399
TitleCryo-EM structure of dCas9 bound to DNA duplex with 10 bp extension
Map data
Sample
  • Complex: Catalytically inactive Cas9 complexed with guide RNA and target DNA duplex
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: Guide RNA
    • DNA: Target DNA, strand 1
    • DNA: Target DNA, strand 2
KeywordsCas9 / DNA BINDING PROTEIN
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA 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, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / 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 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / 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/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes M1 GAS (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsGrassetti AV / Kinman LF / Davis JH
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM144542 United States
National Science Foundation (NSF, United States)2046778 United States
CitationJournal: To Be Published
Title: Engineered Cas9 complexes establish an experimentally grounded benchmark for heterogeneous cryo-EM reconstruction methods
Authors: Grassetti AV / Kinman LF / Davis JH
History
DepositionApr 2, 2026-
Header (metadata) releaseJul 29, 2026-
Map releaseJul 29, 2026-
UpdateJul 29, 2026-
Current statusJul 29, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_76399.png

Downloads & links

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Map

FileDownload / File: emd_76399.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.)
1.12 Å/pix.
x 256 pix.
= 287.168 Å		1.12 Å/pix.
x 256 pix.
= 287.168 Å		1.12 Å/pix.
x 256 pix.
= 287.168 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.12175 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.61029613 - 1.6260664
Average (Standard dev.)-0.0011726943 (±0.033111144)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 287.168 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_76399_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_76399_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 : Catalytically inactive Cas9 complexed with guide RNA and target D...

EntireName: Catalytically inactive Cas9 complexed with guide RNA and target DNA duplex
Components
  • Complex: Catalytically inactive Cas9 complexed with guide RNA and target DNA duplex
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: Guide RNA
    • DNA: Target DNA, strand 1
    • DNA: Target DNA, strand 2

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Supramolecule #1: Catalytically inactive Cas9 complexed with guide RNA and target D...

SupramoleculeName: Catalytically inactive Cas9 complexed with guide RNA and target DNA duplex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus pyogenes M1 GAS (bacteria)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: SNACDKKYSI GLAIGTNSVG WAVITDEYKV PSKKFKVLGN TDRHSIKKNL IGALLFDSGE TAEATRLKRT ARRRYTRRKN RISYLQEIFS NEMAKVDDSF FHRLEESFLV EEDKKHERHP IFGNIVDEVA YHEKYPTIYH LRKKLVDSTD KADLRLIYLA LAHMIKFRGH ...String:
SNACDKKYSI GLAIGTNSVG WAVITDEYKV PSKKFKVLGN TDRHSIKKNL IGALLFDSGE TAEATRLKRT ARRRYTRRKN RISYLQEIFS NEMAKVDDSF FHRLEESFLV EEDKKHERHP IFGNIVDEVA YHEKYPTIYH LRKKLVDSTD KADLRLIYLA LAHMIKFRGH FLIEGDLNPD NSDVDKLFIQ LVQTYNQLFE ENPINASGVD AKAILSARLS KSRRLENLIA QLPGEKKNGL FGNLIALSLG LTPNFKSNFD LAEDAKLQLS KDTYDDDLDN LLAQIGDQYA DLFLAAKNLS DAILLSDILR VNTEITKAPL SASMIKRYDE HHQDLTLLKA LVRQQLPEKY KEIFFDQSKN GYAGYIDGGA SQEEFYKFIK PILEKMDGTE ELLVKLNRED LLRKQRTFDN GSIPHQIHLG ELHAILRRQE DFYPFLKDNR EKIEKILTFR IPYYVGPLAR GNSRFAWMTR KSEETITPWN FEEVVDKGAS AQSFIERMTN FDKNLPNEKV LPKHSLLYEY FTVYNELTKV KYVTEGMRKP AFLSGEQKKA IVDLLFKTNR KVTVKQLKED YFKKIESFDS VEISGVEDRF NASLGTYHDL LKIIKDKDFL DNEENEDILE DIVLTLTLFE DREMIEERLK TYAHLFDDKV MKQLKRRRYT GWGRLSRKLI NGIRDKQSGK TILDFLKSDG FANRNFMQLI HDDSLTFKED IQKAQVSGQG DSLHEHIANL AGSPAIKKGI LQTVKVVDEL VKVMGRHKPE NIVIEMAREN QTTQKGQKNS RERMKRIEEG IKELGSQILK EHPVENTQLQ NEKLYLYYLQ NGRDMYVDQE LDINRLSDYD VDAIVPQSFL KDDSIDNKVL TRSDKNRGKS DNVPSEEVVK KMKNYWRQLL NAKLITQRKF DNLTKAERGG LSELDKAGFI KRQLVETRQI TKHVAQILDS RMNTKYDEND KLIREVKVIT LKSKLVSDFR KDFQFYKVRE INNYHHAHDA YLNAVVGTAL IKKYPKLESE FVYGDYKVYD VRKMIAKSEQ EIGKATAKYF FYSNIMNFFK TEITLANGEI RKRPLIETNG ETGEIVWDKG RDFATVRKVL SMPQVNIVKK TEVQTGGFSK ESILPKRNSD KLIARKKDWD PKKYGGFDSP TVAYSVLVVA KVEKGKSKKL KSVKELLGIT IMERSSFEKN PIDFLEAKGY KEVKKDLIIK LPKYSLFELE NGRKRMLASA GELQKGNELA LPSKYVNFLY LASHYEKLKG SPEDNEQKQL FVEQHKHYLD EIIEQISEFS KRVILADANL DKVLSAYNKH RDKPIREQAE NIIHLFTLTN LGAPAAFKYF DTTIDRKRYT STKEVLDATL IHQSITGLYE TRIDLSQLGG D

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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Macromolecule #2: Guide RNA

MacromoleculeName: Guide RNA / type: rna / ID: 2
SequenceString:
GACGCAUAAA GAUGAGACGC GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUU

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Macromolecule #3: Target DNA, strand 1

MacromoleculeName: Target DNA, strand 1 / type: dna / ID: 3 / Classification: DNA
SequenceString:
TGCTGACGCA TAAAGATGAG ACGCTGGTAA AAAAAAA

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Macromolecule #4: Target DNA, strand 2

MacromoleculeName: Target DNA, strand 2 / type: dna / ID: 4 / Classification: DNA
SequenceString:
TTTTTTTTTA CCAGCGTCTC ATCTTTATGC GTCAGCA

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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

Concentration0.25 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMHEPES
100.0 mMPotassium chloride
5.0 mMMagnesium chloride
1.0 mMDTT
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average exposure time: 3.89 sec. / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 120000
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: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 173173
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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