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- EMDB-24819: Cas9:sgRNA:DNA (S. pyogenes) forming a 3-base-pair R-loop -

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Basic information

Entry
Database: EMDB / ID: EMD-24819
TitleCas9:sgRNA:DNA (S. pyogenes) forming a 3-base-pair R-loop
Map dataLocScale-sharpened map
Sample
  • Complex: Cas9 bound to sgRNA and DNA with 3bp complementarity between RNA and DNA
    • DNA: Non-target DNA strand
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: Single-guide RNA
    • DNA: Target DNA strand
KeywordsDNase / complex / ribonucleoprotein / genome editor / HYDROLASE-RNA-DNA complex
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, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain ...CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 / HNH endonuclease / 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 serotype M1 (bacteria) / synthetic construct (others) / Streptococcus pyogenes (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsCofsky JC / Soczek KM
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Science Foundation (NSF, United States)1817593 United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: CRISPR-Cas9 bends and twists DNA to read its sequence.
Authors: Joshua C Cofsky / Katarzyna M Soczek / Gavin J Knott / Eva Nogales / Jennifer A Doudna /
Abstract: In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that ...In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9-RNA-DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 'reads' snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.
History
DepositionSep 4, 2021-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24819.png

Downloads & links

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Map

FileDownload / File: emd_24819.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocScale-sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 256 pix.
= 268.8 Å		1.05 Å/pix.
x 256 pix.
= 268.8 Å		1.05 Å/pix.
x 256 pix.
= 268.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.09
Minimum - Maximum-14.770318 - 38.077109999999998
Average (Standard dev.)0.09173816 (±0.96560687)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_24819_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened map from Relion

Fileemd_24819_additional_1.map
Annotationunsharpened map from Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: post-processed map from RELION

Fileemd_24819_additional_2.map
Annotationpost-processed map from RELION
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1 from RELION

Fileemd_24819_half_map_1.map
Annotationhalf map 1 from RELION
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2 from RELION

Fileemd_24819_half_map_2.map
Annotationhalf map 2 from RELION
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Cas9 bound to sgRNA and DNA with 3bp complementarity between RNA ...

EntireName: Cas9 bound to sgRNA and DNA with 3bp complementarity between RNA and DNA
Components
  • Complex: Cas9 bound to sgRNA and DNA with 3bp complementarity between RNA and DNA
    • DNA: Non-target DNA strand
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: Single-guide RNA
    • DNA: Target DNA strand

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Supramolecule #1: Cas9 bound to sgRNA and DNA with 3bp complementarity between RNA ...

SupramoleculeName: Cas9 bound to sgRNA and DNA with 3bp complementarity between RNA and DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Streptococcus pyogenes serotype M1 (bacteria)

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Macromolecule #1: Non-target DNA strand

MacromoleculeName: Non-target DNA strand / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.33604 KDa
SequenceString:
(DG)(DA)(DC)(DG)(DC)(DA)(DT)(DA)(DA)(DA) (DG)(DA)(DT)(DG)(DA)(DG)(DA)(DG)(DT)(DT) (DT)(DG)(DG)(DC)(DG)(DA)(DT)(DT)(DA) (DC)

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Macromolecule #4: Target DNA strand

MacromoleculeName: Target DNA strand / type: dna / ID: 4
Details: The 5'-most deoxycytidine is actually N4-cystamine-2'-deoxycytidine
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.190992 KDa
SequenceString:
(DG)(DT)(DA)(DA)(DT)(2YR)(DG)(DC)(DC)(DA) (DT)(DT)(DG)(DT)(DC)(DT)(DC)(DA)(DT) (DC)(DT)(DT)(DT)(DA)(DT)(DG)(DC)(DG)(DT) (DC)

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Macromolecule #2: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes serotype M1 (bacteria)
Molecular weightTheoretical: 158.974125 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAMDKKYSI GLDIGTNSVG WAVITDEYKV PSKKFKVLGN TDRHSIKKNL IGALLFDSGE TAEATRLKRT ARRRYTRRKN RICYLQEIF SNEMAKVDDS FFHRLEESFL VEEDKKHERH PIFGNIVDEV AYHEKYPTIY HLRKKLVDST DKADLRLIYL A LAHMIKFR ...String:
SNAMDKKYSI GLDIGTNSVG WAVITDEYKV PSKKFKVLGN TDRHSIKKNL IGALLFDSGE TAEATRLKRT ARRRYTRRKN RICYLQEIF SNEMAKVDDS FFHRLEESFL VEEDKKHERH PIFGNIVDEV AYHEKYPTIY HLRKKLVDST DKADLRLIYL A LAHMIKFR GHFLIEGDLN PDNSDVDKLF IQLVQTYNQL FEENPINASG VDAKAILSAR LSKSRRLENL IAQLPGEKKN GL FGNLIAL SLGLTPNFKS NFDLAEDAKL QLSKDTYDDD LDNLLAQIGD QYADLFLAAK NLSDAILLSD ILRVNTEITK APL SASMIK RYDEHHQDLT LLKALVRQQL PEKYKEIFFD QSKNGYAGYI DGGASQEEFY KFIKPILEKM DGTEELLVKL NRED LLRKQ RTFDNGSIPH QIHLGELHAI LRRQEDFYPF LKDNREKIEK ILTFRIPYYV GPLARGNSRF AWMTRKSEET ITPWN FEEV VDKGASAQSF IERMTNFDKN LPNEKVLPKH SLLYEYFTVY NELTKVKYVT EGMRKPAFLS GEQKKAIVDL LFKTNR KVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLK TY AHLFDDKVMK QLKRRRYTGW GRLSRKLING IRDKQSGKTI LDFLKSDGFA NRNFMQLIHD DSLTFKEDIQ KAQVSGQG D SLHEHIANLA GSPAIKKGIL QTVKVVDELV KVMGRHKPEN IVIEMARENQ TTQKGQKNSR ERMKRIEEGI KELGSQILK EHPVENTQLQ NEKLYLYYLQ NGRDMYVDQE LDINRLSDYD VDHIVPQSFL KDDSIDNKVL TRSDKNRGKS DNVPSEEVVK KMKNYWRQL LNAKLITQRK FDNLTKAERG GLSELDKAGF IKRQLVETRQ ITKHVAQILD SRMNTKYDEN DKLIREVKVI T LKSKLVSD FRKDFQFYKV REINNYHHAH DAYLNAVVGT ALIKKYPKLE SEFVYGDYKV YDVRKMIAKS EQEIGKATAK YF FYSNIMN FFKTEITLAN GEIRKRPLIE TNGETGEIVW DKGRDFATVR KVLSMPQVNI VKKTEVQTGG FSKESILPKR NSD KLIARK KDWDPKKYGG FDSPTVAYSV LVVAKVEKGK SKKLKSVKEL LGITIMERSS FEKNPIDFLE AKGYKEVKKD LIIK LPKYS LFELENGRKR MLASAGELQK GNELALPSKY VNFLYLASHY EKLKGSPEDN EQKQLFVEQH KHYLDEIIEQ ISEFS KRVI LADANLDKVL SAYNKHRDKP IREQAENIIH LFTLTNLGAP AAFKYFDTTI DRKRYCSTKE VLDATLIHQS ITGLYE TRI DLSQLGGD

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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Macromolecule #3: Single-guide RNA

MacromoleculeName: Single-guide RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 32.79341 KDa
SequenceString:
GGCUGCGUAU UUCUACUCUC AAGUUUUAGA GCUAGAAAUA GCAAGUUAAA AUAAGGCUAG UCCGUUAUCA ACUUGAAAAA GUGGCACCG AGUCGGUGCU UCG

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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
Component:
ConcentrationNameFormula
20.0 mMTris-Cl
200.0 mMpotassium chlorideKCl
100.0 uMdithiothreitol
5.0 mMmagnesium chlorideMgCl2
0.25 %glycerol
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Details: 25mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: models from Cas9:sgRNA:DNA 0bp RNA:DNA matching
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Number images used: 17424
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.0)
FSC plot (resolution estimation)

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