[English] 日本語
Yorodumi
- PDB-7s36: Cas9:sgRNA:DNA (S. pyogenes) with 0 RNA:DNA base pairs, closed-pr... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7s36
TitleCas9:sgRNA:DNA (S. pyogenes) with 0 RNA:DNA base pairs, closed-protein/bent-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 / 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
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: 3.2 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 4, 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

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
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
Water0
1


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

-
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

-
Experimental details

-
Experiment

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

-
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

-
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 FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

-
Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4cryoSPARC3.2CTF correctionpatch ctf
10cryoSPARC3.2initial Euler assignment
11RELION3.1.0final Euler assignment
13RELION3.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18121 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00714345
ELECTRON MICROSCOPYf_angle_d0.9819912
ELECTRON MICROSCOPYf_dihedral_angle_d15.1535835
ELECTRON MICROSCOPYf_chiral_restr0.0482272
ELECTRON MICROSCOPYf_plane_restr0.0062073

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more