[English] 日本語
Yorodumi
- PDB-5vzl: cryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 5vzl
Titlecryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex
Components
  • CRISPR-associated endonuclease Cas9
  • phage anti-CRISPR AcrIIA4
  • single guide RNA (116-MER)
KeywordsImmune System/RNA / anti-CRISPR / Cas9 / CRISPR / gene editing / on-target / off-target / cryo-EM / Immune System-RNA complex
Function / homology
Function and homology information


symbiont-mediated suppression of host CRISPR-cas system / 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, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / : / phage anti-CRISPR AcrIIA4 / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes M1 GAS (bacteria)
unidentified phage (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsJiang, F. / Liu, J.J. / Nogales, E. / Doudna, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Sci Adv / Year: 2017
Title: Disabling Cas9 by an anti-CRISPR DNA mimic.
Authors: Jiyung Shin / Fuguo Jiang / Jun-Jie Liu / Nicolas L Bray / Benjamin J Rauch / Seung Hyun Baik / Eva Nogales / Joseph Bondy-Denomy / Jacob E Corn / Jennifer A Doudna /
Abstract: CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. ...CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.
History
DepositionMay 29, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 26, 2017Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 26, 2017Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2018Group: Author supporting evidence / Data processing ...Author supporting evidence / Data processing / Database references / Experimental preparation
Category: em_sample_support / em_software ...em_sample_support / em_software / pdbx_audit_support / pdbx_database_related
Item: _em_sample_support.grid_type / _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Mar 20, 2019Group: Data collection / Other / Structure summary / Category: cell / struct_keywords
Item: _cell.length_a / _cell.length_b ..._cell.length_a / _cell.length_b / _cell.length_c / _struct_keywords.pdbx_keywords / _struct_keywords.text
Revision 1.3Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.6May 14, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 14, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-8749
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
B: single guide RNA (116-MER)
A: CRISPR-associated endonuclease Cas9
C: phage anti-CRISPR AcrIIA4


Theoretical massNumber of molelcules
Total (without water)207,2483
Polymers207,2483
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17050 Å2
ΔGint-115 kcal/mol
Surface area84820 Å2

-
Components

#1: RNA chain single guide RNA (116-MER)


Mass: 38292.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes M1 GAS (bacteria)
Gene: csn1, cas9, ERS445054_00848 / Production host: Escherichia coli (E. coli)
#2: Protein CRISPR-associated endonuclease Cas9


Mass: 158772.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes M1 GAS (bacteria)
Gene: csn1, cas9, ERS445054_00848 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A0C6FZC2, UniProt: Q99ZW2*PLUS, Hydrolases; Acting on ester bonds
#3: Protein phage anti-CRISPR AcrIIA4


Mass: 10182.073 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified phage (virus) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2D0TCG7*PLUS
Has protein modificationN

-
Experimental details

-
Experiment

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

-
Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4COMPLEXall0RECOMBINANT
2Streptococcus pyogenes M1 GASCOMPLEX#1-#21RECOMBINANT
3unidentified phageCOMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Streptococcus pyogenes M1 GAS (bacteria)160490
32unidentified phage (virus)38018
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 1.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: spyCas9, sgRNA and AcrIIA4 complex
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: blot for 4.5 seconds with force of 12 before plunging

-
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 defocus max: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 900 nm / Calibrated defocus max: 4000 nm / Cs: 0.01 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 46 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

-
Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategoryDetailsFitting-ID
4RELION2CTF correctionCTFFIND4
11RELION2final Euler assignment
21PHENIXmodel refinement1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185000 / Symmetry type: POINT
Atomic model building
IDProtocolSpace
1AB INITIO MODELREAL
2RIGID BODY FITREAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00614414
ELECTRON MICROSCOPYf_angle_d0.92720059
ELECTRON MICROSCOPYf_dihedral_angle_d12.1258452
ELECTRON MICROSCOPYf_chiral_restr0.052356
ELECTRON MICROSCOPYf_plane_restr0.0062153

+
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