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- EMDB-47835: SpCas9 with 17-bp R-loop containing 2 terminal mismatches (State ... -

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Basic information

Entry
Database: EMDB / ID: EMD-47835
TitleSpCas9 with 17-bp R-loop containing 2 terminal mismatches (State II - precleavage)
Map dataprecleavage state
Sample
  • Complex: Cas9-sgRNA-dsDNA complex
    • RNA: sgRNA
    • DNA: DNA (Target Strand)
    • DNA: DNA (Non-target strand)
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
KeywordsType II-A / CRISPR-associated endonuclease / RNA-guided / DNA targeting / Mg2+ dependent / 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, 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 / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...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 / 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 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsKiernan KA / Taylor DW
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
Welch FoundationF-1938-20200401 United States
American Cancer SocietyRSG-21-050-01-DMC United States
CitationJournal: Nat Commun / Year: 2025
Title: Visualization of a multi-turnover Cas9 after product release.
Authors: Kaitlyn A Kiernan / David W Taylor /
Abstract: While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product ...While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Cas9 can eventually be removed from the product by extrinsic factors, such as translocating polymerases, but the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and promote faster enzyme turnover. Using kinetics-guided cryo-EM, we examine the conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.
History
DepositionNov 11, 2024-
Header (metadata) releaseJul 9, 2025-
Map releaseJul 9, 2025-
UpdateJul 9, 2025-
Current statusJul 9, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47835.png

Downloads & links

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Map

FileDownload / File: emd_47835.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprecleavage state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 512 pix.
= 426.598 Å		0.83 Å/pix.
x 512 pix.
= 426.598 Å		0.83 Å/pix.
x 512 pix.
= 426.598 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.4469659 - 0.8704568
Average (Standard dev.)-0.00008737382 (±0.010157779)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 426.5984 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map B

Fileemd_47835_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_47835_half_map_2.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas9-sgRNA-dsDNA complex

EntireName: Cas9-sgRNA-dsDNA complex
Components
  • Complex: Cas9-sgRNA-dsDNA complex
    • RNA: sgRNA
    • DNA: DNA (Target Strand)
    • DNA: DNA (Non-target strand)
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1

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Supramolecule #1: Cas9-sgRNA-dsDNA complex

SupramoleculeName: Cas9-sgRNA-dsDNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: bound to a 17-nt sgRNA containing two terminal mismatches and a 55-bp dsDNA target
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 213.23 KDa

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Macromolecule #1: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 31.335643 KDa
SequenceString:
CGAUAAAGAU GAGACGCGUU UUAGAGCUAG AAAUAGCAAG UUAAAAUAAG GCUAGUCCGU UAUCAACUUG AAAAAGUGGC ACCGAGUCG GUGCUUUU

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Macromolecule #2: DNA (Target Strand)

MacromoleculeName: DNA (Target Strand) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 16.865783 KDa
SequenceString: (DA)(DG)(DC)(DT)(DG)(DA)(DC)(DG)(DT)(DT) (DT)(DG)(DT)(DA)(DC)(DT)(DC)(DC)(DA)(DG) (DC)(DG)(DT)(DC)(DT)(DC)(DA)(DT)(DC) (DT)(DT)(DT)(DA)(DT)(DG)(DC)(DG)(DT)(DC) (DA) (DG)(DC)(DA)(DG)(DA)(DG) ...String:
(DA)(DG)(DC)(DT)(DG)(DA)(DC)(DG)(DT)(DT) (DT)(DG)(DT)(DA)(DC)(DT)(DC)(DC)(DA)(DG) (DC)(DG)(DT)(DC)(DT)(DC)(DA)(DT)(DC) (DT)(DT)(DT)(DA)(DT)(DG)(DC)(DG)(DT)(DC) (DA) (DG)(DC)(DA)(DG)(DA)(DG)(DA)(DT) (DT)(DT)(DC)(DT)(DG)(DC)(DT)

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Macromolecule #3: DNA (Non-target strand)

MacromoleculeName: DNA (Non-target strand) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 17.026959 KDa
SequenceString: (DA)(DG)(DC)(DA)(DG)(DA)(DA)(DA)(DT)(DC) (DT)(DC)(DT)(DG)(DC)(DT)(DG)(DA)(DC)(DG) (DC)(DA)(DT)(DA)(DA)(DA)(DG)(DA)(DT) (DG)(DA)(DG)(DA)(DC)(DG)(DC)(DT)(DG)(DG) (DA) (DG)(DT)(DA)(DC)(DA)(DA) ...String:
(DA)(DG)(DC)(DA)(DG)(DA)(DA)(DA)(DT)(DC) (DT)(DC)(DT)(DG)(DC)(DT)(DG)(DA)(DC)(DG) (DC)(DA)(DT)(DA)(DA)(DA)(DG)(DA)(DT) (DG)(DA)(DG)(DA)(DC)(DG)(DC)(DT)(DG)(DG) (DA) (DG)(DT)(DA)(DC)(DA)(DA)(DA)(DC) (DG)(DT)(DC)(DA)(DG)(DC)(DT)

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Macromolecule #4: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 158.699844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF ...String:
MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA KAILSARLSK SRRLENLIAQ LPGEKKNGLF GN LIALSLG LTPNFKSNFD LAEDAKLQLS KDTYDDDLDN LLAQIGDQYA DLFLAAKNLS DAILLSDILR VNTEITKAPL SAS MIKRYD EHHQDLTLLK ALVRQQLPEK YKEIFFDQSK NGYAGYIDGG ASQEEFYKFI KPILEKMDGT EELLVKLNRE DLLR KQRTF DNGSIPHQIH LGELHAILRR QEDFYPFLKD NREKIEKILT FRIPYYVGPL ARGNSRFAWM TRKSEETITP WNFEE VVDK GASAQSFIER MTNFDKNLPN EKVLPKHSLL YEYFTVYNEL TKVKYVTEGM RKPAFLSGEQ KKAIVDLLFK TNRKVT VKQ LKEDYFKKIE CFDSVEISGV EDRFNASLGT YHDLLKIIKD KDFLDNEENE DILEDIVLTL TLFEDREMIE ERLKTYA HL FDDKVMKQLK RRRYTGWGRL SRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSL H EHIANLAGSP AIKKGILQTV KVVDELVKVM GRHKPENIVI EMARENQTTQ KGQKNSRERM KRIEEGIKEL GSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNA KLITQRKFDN LTKAERGGLS ELDKAGFIKR QLVETRQITK HVAQILDSRM NTKYDENDKL IREVKVITLK S KLVSDFRK DFQFYKVREI NNYHHAHDAY LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YS NIMNFFK TEITLANGEI RKRPLIETNG ETGEIVWDKG RDFATVRKVL SMPQVNIVKK TEVQTGGFSK ESILPKRNSD KLI ARKKDW DPKKYGGFDS PTVAYSVLVV AKVEKGKSKK LKSVKELLGI TIMERSSFEK NPIDFLEAKG YKEVKKDLII KLPK YSLFE LENGRKRMLA SAGELQKGNE LALPSKYVNF LYLASHYEKL KGSPEDNEQK QLFVEQHKHY LDEIIEQISE FSKRV ILAD ANLDKVLSAY NKHRDKPIRE QAENIIHLFT LTNLGAPAAF KYFDTTIDRK RYTSTKEVLD ATLIHQSITG LYETRI DLS QLGGD

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
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 opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
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: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 125923
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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