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- EMDB-31721: Cryo-EM structure of spyCas9-sgRNA-DNA dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-31721
TitleCryo-EM structure of spyCas9-sgRNA-DNA dimer
Map dataCas9 dimer
Sample
  • Complex: Ternary complex of spyCas9 with sgRNA and DNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: RNA (115-MER)
    • DNA: DNA (49-MER)
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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...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 / 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) / Streptococcus pyogenes serotype M1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.26 Å
AuthorsLiu J / Deng P
Funding support China, 2 items
OrganizationGrant numberCountry
Other governmentTsinghua-Peking Joint Center for Life Sciences China
Other governmentBeijing Advanced Innovation Center for Structural Biology China
CitationJournal: Chem Sci / Year: 2021
Title: Nonspecific interactions between SpCas9 and dsDNA sites located downstream of the PAM mediate facilitated diffusion to accelerate target search.
Authors: Mengyi Yang / Ruirui Sun / Pujuan Deng / Yuzhuo Yang / Wenjuan Wang / Jun-Jie Gogo Liu / Chunlai Chen /
Abstract: RNA-guided Cas9 (SpCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how Cas9 locates its target among huge amounts of dsDNAs ...RNA-guided Cas9 (SpCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how Cas9 locates its target among huge amounts of dsDNAs remains elusive. Here, combining biochemical and single-molecule fluorescence assays, we revealed that Cas9 uses both three-dimensional and one-dimensional diffusion to find its target with high efficiency. We further observed surprising apparent asymmetric target search regions flanking PAM sites on dsDNA under physiological salt conditions, which accelerates the target search efficiency of Cas9 by ∼10-fold. Illustrated by a cryo-EM structure of the Cas9/sgRNA/dsDNA dimer, non-specific interactions between DNA ∼8 bp downstream of the PAM site and lysines within residues 1151-1156 of Cas9, especially lys1153, are the key elements to mediate the one-dimensional diffusion of Cas9 and cause asymmetric target search regions flanking the PAM. Disrupting these non-specific interactions, such as mutating these lysines to alanines, diminishes the contribution of one-dimensional diffusion and reduces the target search rate by several times. In addition, low ionic concentrations or mutations on PAM recognition residues that modulate interactions between Cas9 and dsDNA alter apparent asymmetric target search behaviors. Together, our results reveal a unique searching mechanism of Cas9 under physiological salt conditions, and provide important guidance for both and applications of Cas9.
History
DepositionAug 16, 2021-
Header (metadata) releaseAug 17, 2022-
Map releaseAug 17, 2022-
UpdateMar 1, 2023-
Current statusMar 1, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_31721.png

Downloads & links

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Map

FileDownload / File: emd_31721.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas9 dimer
Voxel sizeX=Y=Z: 1.14 Å
Density
Contour LevelBy AUTHOR: 0.217
Minimum - Maximum-0.4338241 - 0.8681433
Average (Standard dev.)-0.001109847 (±0.033154387)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 364.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Ternary complex of spyCas9 with sgRNA and DNA

EntireName: Ternary complex of spyCas9 with sgRNA and DNA
Components
  • Complex: Ternary complex of spyCas9 with sgRNA and DNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: RNA (115-MER)
    • DNA: DNA (49-MER)

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Supramolecule #1: Ternary complex of spyCas9 with sgRNA and DNA

SupramoleculeName: Ternary complex of spyCas9 with sgRNA and DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus pyogenes (bacteria)

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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: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes serotype M1 (bacteria)
Molecular weightTheoretical: 158.111234 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL QEIFSNEMA KVDDSFFHRL EESFLVEEDK KHERHPIFGN IVDEVAYHEK YPTIYHLRKK LVDSTDKADL RLIYLALAHM I KFRGHFLI ...String:
KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL QEIFSNEMA KVDDSFFHRL EESFLVEEDK KHERHPIFGN IVDEVAYHEK YPTIYHLRKK LVDSTDKADL RLIYLALAHM I KFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN LI ALSLGLT PNFKSNFDLA EDAKLQLSKD TYDDDLDNLL AQIGDQYADL FLAAKNLSDA ILLSDILRVN TEITKAPLSA SMI KRYDEH HQDLTLLKAL VRQQLPEKYK EIFFDQSKNG YAGYIDGGAS QEEFYKFIKP ILEKMDGTEE LLVKLNREDL LRKQ RTFDN GSIPHQIHLG ELHAILRRQE DFYPFLKDNR EKIEKILTFR IPYYVGPLAR GNSRFAWMTR KSEETITPWN FEEVV DKGA SAQSFIERMT NFDKNLPNEK VLPKHSLLYE YFTVYNELTK VKYVTEGMRK PAFLSGEQKK AIVDLLFKTN RKVTVK QLK EDYFKKIECF DSVEISGVED RFNASLGTYH DLLKIIKDKD FLDNEENEDI LEDIVLTLTL FEDREMIEER LKTYAHL FD DKVMKQLKRR RYTGWGRLSR KLINGIRDKQ SGKTILDFLK SDGFANRNFM QLIHDDSLTF KEDIQKAQVS GQGDSLHE H IANLAGSPAI KKGILQTVKV VDELVKVMGR HKPENIVIEM ARENQTTQKG QKNSRERMKR IEEGIKELGS QILKEHPVE NTQLQNEKLY LYYLQNGRDM YVDQELDINR LSDYDVDHIV PQSFLKDDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY WRQLLNAKL ITQRKFDNLT KAERGGLSEL DKAGFIKRQL VETRQITKHV AQILDSRMNT KYDENDKLIR EVKVITLKSK L VSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS NI MNFFKTE ITLANGEIRK RPLIETNGET GEIVWDKGRD FATVRKVLSM PQVNIVKKTE VQTGGFSKES ILPKRNSDKL IAR KKDWDP KKYGGFDSPT VAYSVLVVAK VEKGKSKKLK SVKELLGITI MERSSFEKNP IDFLEAKGYK EVKKDLIIKL PKYS LFELE NGRKRMLASA GELQKGNELA LPSKYVNFLY LASHYEKLKG SPEDNEQKQL FVEQHKHYLD EIIEQISEFS KRVIL ADAN LDKVLSAYNK HRDKPIREQA ENIIHLFTLT NLGAPAAFKY FDTTIDRKRY TSTKEVLDAT LIHQSITGLY ETRIDL SQ

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Macromolecule #2: RNA (115-MER)

MacromoleculeName: RNA (115-MER) / type: rna / ID: 2 / Number of copies: 2
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 37.13709 KDa
SequenceString:
GCGCAUAAAG AUGAGACGCG UUUUAGAGCU AUGCUGUUUU GAAAAAAACA GCAUAGCAAG UUAAAAUAAG GCUAGUCCGU UAUCAACUU GAAAAAGUGG CACCGAGUCG GUGCUU

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Macromolecule #3: DNA (49-MER)

MacromoleculeName: DNA (49-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 15.040633 KDa
SequenceString:
(DA)(DT)(DG)(DA)(DG)(DA)(DC)(DG)(DC)(DT) (DG)(DG)(DC)(DG)(DA)(DT)(DT)(DA)(DG)(DC) (DT)(DA)(DA)(DT)(DC)(DG)(DC)(DC)(DA) (DG)(DC)(DG)(DT)(DC)(DT)(DC)(DA)(DT)(DC) (DT) (DT)(DT)(DA)(DT)(DG)(DC)(DG)(DC) (DC)

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5f9r

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.26 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 150080

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