[English] 日本語
Yorodumi
- EMDB-0583: Conformational states of Cas9-sgRNA-DNA ternary complex in the pr... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-0583
TitleConformational states of Cas9-sgRNA-DNA ternary complex in the presence of magnesium
Map dataprimary map
Sample
  • Complex: Cas9-sgRNA-DNA ternary complex
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: single guide RNA
    • DNA: 5' product of target strand DNA
    • DNA: non-target strand DNA
    • DNA: 3' product of target strand DNA
KeywordsCRISPR / Cas9 / Nuclease / Genome editing / HYDROLASE / 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 / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...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 / HNH endonuclease / CRISPR-associated endonuclease Cas9 / 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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsZhu X / Clarke R
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)097042 United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)081534 United States
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.
Authors: Xing Zhu / Ryan Clarke / Anupama K Puppala / Sagar Chittori / Alan Merk / Bradley J Merrill / Miljan Simonović / Sriram Subramaniam /
Abstract: The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures ...The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg. In the precatalytic state, Cas9 adopts the 'checkpoint' conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9.
History
DepositionFeb 17, 2019-
Header (metadata) releaseMay 22, 2019-
Map releaseJul 10, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6o0x
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0583.png

Downloads & links

-
Map

FileDownload / File: emd_0583.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprimary map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 304 pix.
= 254.144 Å		0.84 Å/pix.
x 304 pix.
= 254.144 Å		0.84 Å/pix.
x 304 pix.
= 254.144 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.836 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.6366258 - 1.3570119
Average (Standard dev.)-0.0010377981 (±0.043065052)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 254.14401 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8360.8360.836
M x/y/z304304304
origin x/y/z0.0000.0000.000
length x/y/z254.144254.144254.144
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS304304304
D min/max/mean-0.6371.357-0.001

-
Supplemental data

-
Sample components

-
Entire : Cas9-sgRNA-DNA ternary complex

EntireName: Cas9-sgRNA-DNA ternary complex
Components
  • Complex: Cas9-sgRNA-DNA ternary complex
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: single guide RNA
    • DNA: 5' product of target strand DNA
    • DNA: non-target strand DNA
    • DNA: 3' product of target strand DNA

-
Supramolecule #1: Cas9-sgRNA-DNA ternary complex

SupramoleculeName: Cas9-sgRNA-DNA ternary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus pyogenes (bacteria)

-
Macromolecule #1: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 158.986109 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAASMDKKYS IGLDIGTNSV GWAVITDEYK VPSKKFKVLG NTDRHSIKKN LIGALLFDSG ETAEATRLKR TARRRYTRRK NRICYLQEI FSNEMAKVDD SFFHRLEESF LVEEDKKHER HPIFGNIVDE VAYHEKYPTI YHLRKKLVDS TDKADLRLIY L ALAHMIKF ...String:
GAASMDKKYS IGLDIGTNSV GWAVITDEYK VPSKKFKVLG NTDRHSIKKN LIGALLFDSG ETAEATRLKR TARRRYTRRK NRICYLQEI FSNEMAKVDD SFFHRLEESF LVEEDKKHER HPIFGNIVDE VAYHEKYPTI YHLRKKLVDS TDKADLRLIY L ALAHMIKF RGHFLIEGDL NPDNSDVDKL FIQLVQTYNQ LFEENPINAS GVDAKAILSA RLSKSRRLEN LIAQLPGEKK NG LFGNLIA LSLGLTPNFK SNFDLAEDAK LQLSKDTYDD DLDNLLAQIG DQYADLFLAA KNLSDAILLS DILRVNTEIT KAP LSASMI KRYDEHHQDL TLLKALVRQQ LPEKYKEIFF DQSKNGYAGY IDGGASQEEF YKFIKPILEK MDGTEELLVK LNRE DLLRK QRTFDNGSIP HQIHLGELHA ILRRQEDFYP FLKDNREKIE KILTFRIPYY VGPLARGNSR FAWMTRKSEE TITPW NFEE VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTN RKV TVKQLKEDYF KKIECFDSVE ISGVEDRFNA SLGTYHDLLK IIKDKDFLDN EENEDILEDI VLTLTLFEDR EMIEERL KT YAHLFDDKVM KQLKRRRYTG WGRLSRKLIN GIRDKQSGKT ILDFLKSDGF ANRNFMQLIH DDSLTFKEDI QKAQVSGQ G DSLHEHIANL AGSPAIKKGI LQTVKVVDEL VKVMGRHKPE NIVIEMAREN QTTQKGQKNS RERMKRIEEG IKELGSQIL KEHPVENTQL QNEKLYLYYL QNGRDMYVDQ ELDINRLSDY DVDHIVPQSF LKDDSIDNKV LTRSDKNRGK SDNVPSEEVV KKMKNYWRQ LLNAKLITQR KFDNLTKAER GGLSELDKAG FIKRQLVETR QITKHVAQIL DSRMNTKYDE NDKLIREVKV I TLKSKLVS DFRKDFQFYK VREINNYHHA HDAYLNAVVG TALIKKYPKL ESEFVYGDYK VYDVRKMIAK SEQEIGKATA KY FFYSNIM NFFKTEITLA NGEIRKRPLI ETNGETGEIV WDKGRDFATV RKVLSMPQVN IVKKTEVQTG GFSKESILPK RNS DKLIAR KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKE LLGITIMERS SFEKNPIDFL EAKGYKEVKK DLII KLPKY SLFELENGRK RMLASAGELQ KGNELALPSK YVNFLYLASH YEKLKGSPED NEQKQLFVEQ HKHYLDEIIE QISEF SKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLY ETR IDLSQLGGD

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

-
Macromolecule #2: single guide RNA

MacromoleculeName: single guide RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 33.059699 KDa
SequenceString:
GGGGCGCAUA AAGAUGAGAC GCGUUUUAGA GCUAGAAAUA GCAAGUUAAA AUAAGGCUAG UCCGUUAUCA ACUUGAAAAA GUGGCACCG AGUCGGUGCU UGC

-
Macromolecule #3: 5' product of target strand DNA

MacromoleculeName: 5' product of target strand DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.936575 KDa
SequenceString:
(DC)(DT)(DA)(DA)(DT)(DC)(DG)(DC)(DC)(DA) (DG)(DC)(DG)

-
Macromolecule #4: non-target strand DNA

MacromoleculeName: non-target strand DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.452001 KDa
SequenceString:
(DC)(DC)(DA)(DG)(DT)(DG)(DC)(DG)(DT)(DA) (DG)(DG)(DC)(DG)(DC)(DA)(DT)(DA)(DA)(DA) (DG)(DA)(DT)(DG)(DA)(DG)(DA)(DC)(DG) (DC)(DT)(DG)(DG)(DC)(DG)(DA)(DT)(DT)(DA) (DG)

-
Macromolecule #5: 3' product of target strand DNA

MacromoleculeName: 3' product of target strand DNA / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.194269 KDa
SequenceString:
(DT)(DC)(DT)(DC)(DA)(DT)(DC)(DT)(DT)(DT) (DA)(DT)(DG)(DC)(DG)(DC)(DC)(DT)(DA)(DC) (DG)(DC)(DA)(DC)(DT)(DG)(DG)

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 7.5
GridDetails: unspecified
VitrificationCryogen name: ETHANE

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 73.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 30513
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

+
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