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- EMDB-41775: SpG Cas9 with NGC PAM DNA target -

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Basic information

Entry
Database: EMDB / ID: EMD-41775
TitleSpG Cas9 with NGC PAM DNA target
Map data
Sample
  • Complex: SpG Cas9 with NGG PAM target
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: RNA (98-MER)
    • DNA: DNA (5'-D(P*CP*GP*TP*TP*TP*GP*TP*AP*CP*TP*GP*CP*AP*GP*CP*G)-3')
    • DNA: DNA (5'-D(P*TP*CP*TP*CP*AP*TP*CP*TP*TP*TP*AP*TP*GP*CP*GP*TP*C)-3')
    • DNA: DNA (5'-D(P*TP*GP*CP*AP*GP*TP*AP*CP*AP*AP*AP*CP*G)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsSpG-Cas9 / CRISPR / Cas9 / IMMUNE SYSTEM / R-loop / IMMUNE SYSTEM-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 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsBravo JPK / Hibshman GN / 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
DepositionAug 28, 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_41775.png

Downloads & links

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Map

FileDownload / File: emd_41775.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.
= 319.872 Å		0.83 Å/pix.
x 384 pix.
= 319.872 Å		0.83 Å/pix.
x 384 pix.
= 319.872 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.833 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.434
Minimum - Maximum-0.9451912 - 2.0036352
Average (Standard dev.)0.0015753886 (±0.04521346)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 319.872 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_41775_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_41775_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 : SpG Cas9 with NGG PAM target

EntireName: SpG Cas9 with NGG PAM target
Components
  • Complex: SpG Cas9 with NGG PAM target
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: RNA (98-MER)
    • DNA: DNA (5'-D(P*CP*GP*TP*TP*TP*GP*TP*AP*CP*TP*GP*CP*AP*GP*CP*G)-3')
    • DNA: DNA (5'-D(P*TP*CP*TP*CP*AP*TP*CP*TP*TP*TP*AP*TP*GP*CP*GP*TP*C)-3')
    • DNA: DNA (5'-D(P*TP*GP*CP*AP*GP*TP*AP*CP*AP*AP*AP*CP*G)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: SpG Cas9 with NGG PAM target

SupramoleculeName: SpG Cas9 with NGG PAM target / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
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: 160.860484 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKRTADGSEF ESPKKKRKVD KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRR YTRRKNRICY LQEIFSNEMA KVDDSFFHRL EESFLVEEDK KHERHPIFGN IVDEVAYHEK YPTIYHLRKK L VDSTDKAD ...String:
MKRTADGSEF ESPKKKRKVD KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRR YTRRKNRICY LQEIFSNEMA KVDDSFFHRL EESFLVEEDK KHERHPIFGN IVDEVAYHEK YPTIYHLRKK L VDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RL ENLIAQL PGEKKNGLFG NLIALSLGLT PNFKSNFDLA EDAKLQLSKD TYDDDLDNLL AQIGDQYADL FLAAKNLSDA ILL SDILRV NTEITKAPLS ASMIKRYDEH HQDLTLLKAL VRQQLPEKYK EIFFDQSKNG YAGYIDGGAS QEEFYKFIKP ILEK MDGTE ELLVKLNRED LLRKQRTFDN GSIPHQIHLG ELHAILRRQE DFYPFLKDNR EKIEKILTFR IPYYVGPLAR GNSRF AWMT RKSEETITPW NFEEVVDKGA SAQSFIERMT NFDKNLPNEK VLPKHSLLYE YFTVYNELTK VKYVTEGMRK PAFLSG EQK KAIVDLLFKT NRKVTVKQLK EDYFKKIECF DSVEISGVED RFNASLGTYH DLLKIIKDKD FLDNEENEDI LEDIVLT LT LFEDREMIEE RLKTYAHLFD DKVMKQLKRR RYTGWGRLSR KLINGIRDKQ SGKTILDFLK SDGFANRNFM QLIHDDSL T FKEDIQKAQV SGQGDSLHEH IANLAGSPAI KKGILQTVKV VDELVKVMGR HKPENIVIEM ARENQTTQKG QKNSRERMK RIEEGIKELG SQILKEHPVE NTQLQNEKLY LYYLQNGRDM YVDQELDINR LSDYDVDHIV PQSFLKDDSI DNKVLTRSDK NRGKSDNVP SEEVVKKMKN YWRQLLNAKL ITQRKFDNLT KAERGGLSEL DKAGFIKRQL VETRQITKHV AQILDSRMNT K YDENDKLI REVKVITLKS KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK MI AKSEQEI GKATAKYFFY SNIMNFFKTE ITLANGEIRK RPLIETNGET GEIVWDKGRD FATVRKVLSM PQVNIVKKTE VQT GGFSKE SILPKRNSDK LIARKKDWDP KKYGGFLWPT VAYSVLVVAK VEKGKSKKLK SVKELLGITI MERSSFEKNP IDFL EAKGY KEVKKDLIIK LPKYSLFELE NGRKRMLASA KQLQKGNELA LPSKYVNFLY LASHYEKLKG SPEDNEQKQL FVEQH KHYL DEIIEQISEF SKRVILADAN LDKVLSAYNK HRDKPIREQA ENIIHLFTLT NLGAPAAFKY FDTTIDRKQY RSTKEV LDA TLIHQSITGL YETRIDLSQL GG

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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Macromolecule #2: RNA (98-MER)

MacromoleculeName: RNA (98-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 31.7029 KDa
SequenceString:
GACGCAUAAA GAUGAGACGC GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAG UCGGUGCUU

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Macromolecule #3: DNA (5'-D(P*CP*GP*TP*TP*TP*GP*TP*AP*CP*TP*GP*CP*AP*GP*CP*G)-3')

MacromoleculeName: DNA (5'-D(P*CP*GP*TP*TP*TP*GP*TP*AP*CP*TP*GP*CP*AP*GP*CP*G)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.905176 KDa
SequenceString:
(DC)(DG)(DT)(DT)(DT)(DG)(DT)(DA)(DC)(DT) (DG)(DC)(DA)(DG)(DC)(DG)

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Macromolecule #4: DNA (5'-D(P*TP*CP*TP*CP*AP*TP*CP*TP*TP*TP*AP*TP*GP*CP*GP*TP*C)-3')

MacromoleculeName: DNA (5'-D(P*TP*CP*TP*CP*AP*TP*CP*TP*TP*TP*AP*TP*GP*CP*GP*TP*C)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.119319 KDa
SequenceString:
(DT)(DC)(DT)(DC)(DA)(DT)(DC)(DT)(DT)(DT) (DA)(DT)(DG)(DC)(DG)(DT)(DC)

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Macromolecule #5: DNA (5'-D(P*TP*GP*CP*AP*GP*TP*AP*CP*AP*AP*AP*CP*G)-3')

MacromoleculeName: DNA (5'-D(P*TP*GP*CP*AP*GP*TP*AP*CP*AP*AP*AP*CP*G)-3')
type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.984625 KDa
SequenceString:
(DT)(DG)(DC)(DA)(DG)(DT)(DA)(DC)(DA)(DA) (DA)(DC)(DG)

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 5 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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 reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 27946
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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