[English] 日本語
Yorodumi
- PDB-7vw3: Cryo-EM structure of SaCas9-sgRNA-DNA ternary complex -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7vw3
TitleCryo-EM structure of SaCas9-sgRNA-DNA ternary complex
Components
  • CRISPR-associated endonuclease Cas9
  • Non-target DNA strand
  • Target DNA strand
  • single-guide RNA
KeywordsDNA BINDING PROTEIN/RNA/DNA / CRISPR-Cas9 / genome editing / DNA cleavage / DNA BINDING PROTEIN / DNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 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, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease ...: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / 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
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsDu, W.H. / Qian, J.Q. / Huang, Q.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971377 China
National Natural Science Foundation of China (NSFC)31671386 China
CitationJournal: Int J Mol Sci / Year: 2023
Title: Full-Length Model of SaCas9-sgRNA-DNA Complex in Cleavage State.
Authors: Wenhao Du / Haixia Zhu / Jiaqiang Qian / Dongmei Xue / Sen Zheng / Qiang Huang /
Abstract: Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined ... Cas9 (SaCas9) is a widely used genome editing tool. Understanding its molecular mechanisms of DNA cleavage could effectively guide the engineering optimization of this system. Here, we determined the first cryo-electron microscopy structure of the SaCas9-sgRNA-DNA ternary complex. This structure reveals that the HNH nuclease domain is tightly bound to the cleavage site of the target DNA strand, and is in close contact with the WED and REC domains. Moreover, it captures the complete structure of the sgRNA, including the previously unresolved stem-loop 2. Based on this structure, we build a full-length model for the ternary complex in cleavage state. This model enables identification of the residues for the interactions between the HNH domain and the WED and REC domains. Moreover, we found that the stem-loop 2 of the sgRNA tightly binds to the PI and RuvC domains and may also regulate the position shift of the RuvC domain. Further mutagenesis and molecular dynamics simulations supported the idea that the interactions of the HNH domain with the WED and REC domains play an important role in the DNA cleavage. Thus, this study provides new mechanistic insights into the DNA cleavage of SaCas9 and is also useful for guiding the future engineering of SaCas9-mediated gene editing systems.
History
DepositionNov 9, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 15, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
B: single-guide RNA
C: Target DNA strand
D: Non-target DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)178,1857
Polymers178,1134
Non-polymers733
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area23810 Å2
ΔGint-177 kcal/mol
Surface area74180 Å2

-
Components

#1: Protein CRISPR-associated endonuclease Cas9 / SaCas9


Mass: 123940.508 Da / Num. of mol.: 1 / Mutation: D10A N580A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: cas9 / Production host: Escherichia coli (E. coli)
References: UniProt: J7RUA5, Hydrolases; Acting on ester bonds
#2: RNA chain single-guide RNA / sgRNA


Mass: 33217.531 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Target DNA strand / TS


Mass: 12372.006 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Non-target DNA strand / NTS


Mass: 8582.533 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

-
Experimental details

-
Experiment

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

-
Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1SaCas9-sgRNA-target DNA ternary complexCOMPLEX#1-#40MULTIPLE SOURCES
2SaCas9COMPLEX#11RECOMBINANT
3sgRNA-target DNACOMPLEX#2-#41SYNTHETIC
Molecular weightValue: 0.198 MDa / Experimental value: NO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 20mM Tris-Cl (pH 7.5), 100mM KCl, 5mM MgCl2, 1mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
120.0 mMtrihydroxymethyl aminomethaneTris1
2100.0 mMpotassium chlorideKCl1
35.0 mMmagnesium chlorideMgCl21
41.0 mMdithiothreitolDTT1
SpecimenConc.: 0.22 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: blot for 4 seconds before pluging
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1800 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 38 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 10389
Image scansWidth: 5760 / Height: 4096

-
Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7Rosetta3.6model fitting
10RELION3.0.8final Euler assignment
11RELION3.0.8classification
12RELION3.0.83D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3872337
Details: We used the program PARSED developed in our previous study to pick the complex particles from the micrographs(Yao R, et al. Bioinformatics. 2020,36:1252-1259).
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 217173 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15CZZ

5czz

15CZZ1PDBexperimental model
25Y36

5y36

15Y362PDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 236.8 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004912889
ELECTRON MICROSCOPYf_angle_d0.747818141
ELECTRON MICROSCOPYf_chiral_restr0.04622085
ELECTRON MICROSCOPYf_plane_restr0.0051703
ELECTRON MICROSCOPYf_dihedral_angle_d21.51373170

+
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://pdbjlvh1.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