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- EMDB-41083: SpRY-Cas9:gRNA complex targeting TAC PAM DNA with 18 bp R-loop -

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Basic information

Entry
Database: EMDB / ID: EMD-41083
TitleSpRY-Cas9:gRNA complex targeting TAC PAM DNA with 18 bp R-loop
Map data
Sample
  • Complex: Ternary complex of SpRY-Cas9 with gRNA and NAC PAM DNA after one minute of DNA incubation with 13 bp R-loop
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: gRNA
    • DNA: TS
    • DNA: NTS
  • Ligand: MAGNESIUM ION
KeywordsSpRY-Cas9 / CRISPR / Cas9 / IMMUNE SYSTEM / R-loop
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 (bacteria) / Escherichia phage Lambda (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsHibshman GN / Bravo JPK / Taylor DW
Funding support United States, 2 items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Nat Commun / Year: 2024
Title: Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.
Authors: Grace N Hibshman / Jack P K Bravo / Matthew M Hooper / Tyler L Dangerfield / Hongshan Zhang / Ilya J Finkelstein / Kenneth A Johnson / David W Taylor /
Abstract: CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable ...CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.
History
DepositionJun 19, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41083.png

Downloads & links

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Map

FileDownload / File: emd_41083.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 319.949 Å		0.83 Å/pix.
x 384 pix.
= 319.949 Å		0.83 Å/pix.
x 384 pix.
= 319.949 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: 0.0634
Minimum - Maximum-0.24499808 - 0.5104091
Average (Standard dev.)0.00017491549 (±0.010318569)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 319.9488 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_41083_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41083_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 : Ternary complex of SpRY-Cas9 with gRNA and NAC PAM DNA after one ...

EntireName: Ternary complex of SpRY-Cas9 with gRNA and NAC PAM DNA after one minute of DNA incubation with 13 bp R-loop
Components
  • Complex: Ternary complex of SpRY-Cas9 with gRNA and NAC PAM DNA after one minute of DNA incubation with 13 bp R-loop
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: gRNA
    • DNA: TS
    • DNA: NTS
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of SpRY-Cas9 with gRNA and NAC PAM DNA after one ...

SupramoleculeName: Ternary complex of SpRY-Cas9 with gRNA and NAC PAM DNA after one minute of DNA incubation with 13 bp R-loop
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 211.54 KDa

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Macromolecule #1: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 159.149406 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE RTRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF ...String:
EDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE RTRLKRTARR 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 ESIRPKRNSD KLI ARKKDW DPKKYGGFLW PTVAYSVLVV AKVEKGKSKK LKSVKELLGI TIMERSSFEK NPIDFLEAKG YKEVKKDLII KLPK YSLFE LENGRKRMLA SAKQLQKGNE LALPSKYVNF LYLASHYEKL KGSPEDNEQK QLFVEQHKHY LDEIIEQISE FSKRV ILAD ANLDKVLSAY NKHRDKPIRE QAENIIHLFT LTRLGAPRAF KYFDTTIDPK QYRSTKEVLD ATLIHQSITG LYETRI DLS QLGGDG

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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Macromolecule #2: gRNA

MacromoleculeName: gRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 31.028494 KDa
SequenceString:
CGCAUAAAGA UGAGACGCGU UUUAGAGCUA GAAAUAGCAA GUUAAAAUAA GGCUAGUCCG UUAUCAACUU GAAAAAGUGG CACCGAGUC GGUGCUU

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Macromolecule #3: TS

MacromoleculeName: TS / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia phage Lambda (virus)
Molecular weightTheoretical: 16.92082 KDa
SequenceString: (DA)(DG)(DC)(DT)(DG)(DA)(DC)(DG)(DT)(DT) (DT)(DG)(DT)(DA)(DC)(DT)(DG)(DT)(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)(DG)(DT)(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 #4: NTS

MacromoleculeName: NTS / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia phage Lambda (virus)
Molecular weightTheoretical: 16.970938 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)(DA)(DC) (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)(DA)(DC) (DA) (DG)(DT)(DA)(DC)(DA)(DA)(DA)(DC) (DG)(DT)(DC)(DA)(DG)(DC)(DT)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 3 / 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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 90380
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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