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Open data
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Basic information
Entry | ![]() | |||||||||
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Title | SpG Cas9 with NGC PAM DNA target | |||||||||
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![]() | SpG-Cas9 / CRISPR / Cas9 / IMMUNE SYSTEM / R-loop / IMMUNE SYSTEM-RNA-DNA complex | |||||||||
Function / homology | ![]() 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 | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.56 Å | |||||||||
![]() | Bravo JPK / Hibshman GN / Taylor DW | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 204 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 18.5 KB 18.5 KB | Display Display | ![]() |
Images | ![]() | 34.9 KB | ||
Filedesc metadata | ![]() | 6.8 KB | ||
Others | ![]() ![]() | 200.2 MB 200.2 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8tzzMC ![]() 8spqC ![]() 8sqhC ![]() 8srsC ![]() 8t6oC ![]() 8t6pC ![]() 8t6sC ![]() 8t6tC ![]() 8t6xC ![]() 8t6yC ![]() 8t76C ![]() 8t77C ![]() 8t78C ![]() 8t79C ![]() 8t7sC ![]() 8u3yC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 0.833 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #1
File | emd_41775_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_41775_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : SpG Cas9 with NGG PAM target
Entire | Name: SpG Cas9 with NGG PAM target |
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Components |
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-Supramolecule #1: SpG Cas9 with NGG PAM target
Supramolecule | Name: SpG Cas9 with NGG PAM target / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 211.54 KDa |
-Macromolecule #1: CRISPR-associated endonuclease Cas9/Csn1
Macromolecule | Name: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 160.860484 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: RNA (98-MER)
Macromolecule | Name: RNA (98-MER) / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 31.7029 KDa |
Sequence | String: GACGCAUAAA GAUGAGACGC GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAG UCGGUGCUU |
-Macromolecule #3: DNA (5'-D(P*CP*GP*TP*TP*TP*GP*TP*AP*CP*TP*GP*CP*AP*GP*CP*G)-3')
Macromolecule | Name: 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 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 4.905176 KDa |
Sequence | String: (DC)(DG)(DT)(DT)(DT)(DG)(DT)(DA)(DC)(DT) (DG)(DC)(DA)(DG)(DC)(DG) |
-Macromolecule #4: DNA (5'-D(P*TP*CP*TP*CP*AP*TP*CP*TP*TP*TP*AP*TP*GP*CP*GP*TP*C)-3')
Macromolecule | Name: 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 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 5.119319 KDa |
Sequence | String: (DT)(DC)(DT)(DC)(DA)(DT)(DC)(DT)(DT)(DT) (DA)(DT)(DG)(DC)(DG)(DT)(DC) |
-Macromolecule #5: DNA (5'-D(P*TP*GP*CP*AP*GP*TP*AP*CP*AP*AP*AP*CP*G)-3')
Macromolecule | Name: 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 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 3.984625 KDa |
Sequence | String: (DT)(DG)(DC)(DA)(DG)(DT)(DA)(DC)(DA)(DA) (DA)(DC)(DG) |
-Macromolecule #6: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #7: water
Macromolecule | Name: water / type: ligand / ID: 7 / Number of copies: 5 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ![]() ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination 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 model | Type of model: NONE |
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Final reconstruction | Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 27946 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |