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- PDB-7s3h: Cas9:sgRNA:DNA (S. pyogenes) with 0 RNA:DNA base pairs, open-prot... -

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Open data


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Basic information

Entry
Database: PDB / ID: 7s3h
TitleCas9:sgRNA:DNA (S. pyogenes) with 0 RNA:DNA base pairs, open-protein/linear-DNA conformation
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • Non-target DNA strand
  • Single-guide RNA
  • Target DNA strand
KeywordsHYDROLASE/RNA/DNA / DNase / complex / ribonucleoprotein / genome editor / HYDROLASE-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
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
synthetic construct (others)
Streptococcus pyogenes (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsCofsky, J.C. / Soczek, K.M. / Knott, G.J. / Nogales, E. / Doudna, J.A.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Science Foundation (NSF, United States)1817593 United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: CRISPR-Cas9 bends and twists DNA to read its sequence.
Authors: Joshua C Cofsky / Katarzyna M Soczek / Gavin J Knott / Eva Nogales / Jennifer A Doudna /
Abstract: In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that ...In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9-RNA-DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 'reads' snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.
History
DepositionSep 6, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 20, 2022Provider: repository / Type: Initial release
Revision 1.1May 4, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 6, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
N: Non-target DNA strand
P: CRISPR-associated endonuclease Cas9/Csn1
R: Single-guide RNA
T: Target DNA strand


Theoretical massNumber of molelcules
Total (without water)210,2674
Polymers210,2674
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: DNA chain Non-target DNA strand


Mass: 9314.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158974.125 Da / Num. of mol.: 1 / Mutation: T1337C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#3: RNA chain Single-guide RNA


Mass: 32787.355 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: DNA chain Target DNA strand


Mass: 9190.992 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The 5'-most deoxycytidine is actually N4-cystamine 2'-deoxycytidine
Source: (synth.) synthetic construct (others)
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cas9 bound to sgRNA and DNA with 0bp complementarity between RNA and DNA
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Streptococcus pyogenes serotype M1 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-Cl1
2200 mMpotassium chlorideKCl1
3100 uMdithiothreitol1
45 mMmagnesium chlorideMgCl21
50.25 %glycerol1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 25mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2particle selection
2SerialEMimage acquisition
4cryoSPARC3.2CTF correctionpatch ctf
10cryoSPARC3.2initial Euler assignment
11cryoSPARC3.2final Euler assignment
13cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 545450 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00710292
ELECTRON MICROSCOPYf_angle_d1.0714187
ELECTRON MICROSCOPYf_dihedral_angle_d16.9244113
ELECTRON MICROSCOPYf_chiral_restr0.051606
ELECTRON MICROSCOPYf_plane_restr0.0061542

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