[English] 日本語
Yorodumi
- EMDB-14501: SpCas9 bound to 18-nucleotide complementary DNA substrate in the ... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-14501
TitleSpCas9 bound to 18-nucleotide complementary DNA substrate in the checkpoint state
Map data
Sample
  • Complex: Ternary complex of SpCas9-sgRNA bound to a 18-nucleotide complementary DNA substrate in the inactive state
    • RNA: sgRNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • DNA: Target strand of 18-nucleotide complementary DNA substrate
    • DNA: Non-target strand of 18-nucleotide complementary DNA substrate
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM ION
  • Ligand: water
KeywordsCRISPR / Cas9 / R-loop / substrate binding / off-target / HYDROLASE
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
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.54 Å
AuthorsPacesa M / Jinek M
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_182567 Switzerland
CitationJournal: Nature / Year: 2022
Title: R-loop formation and conformational activation mechanisms of Cas9.
Authors: Martin Pacesa / Luuk Loeff / Irma Querques / Lena M Muckenfuss / Marta Sawicka / Martin Jinek /
Abstract: Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications, yet its precise ...Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.
History
DepositionMar 4, 2022-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

-
Structure visualization

Supplemental images

emd_14501.png

Downloads & links

-
Map

FileDownload / File: emd_14501.map.gz / Format: CCP4 / Size: 488.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.45 Å/pix.
x 504 pix.
= 226.8 Å		0.45 Å/pix.
x 504 pix.
= 226.8 Å		0.45 Å/pix.
x 504 pix.
= 226.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.45 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.022
Minimum - Maximum-0.089672565 - 0.22747503
Average (Standard dev.)0.0009289633 (±0.0060837837)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions504504504
Spacing504504504
CellA=B=C: 226.79999 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: #2

Fileemd_14501_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: #1

Fileemd_14501_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : Ternary complex of SpCas9-sgRNA bound to a 18-nucleotide compleme...

EntireName: Ternary complex of SpCas9-sgRNA bound to a 18-nucleotide complementary DNA substrate in the inactive state
Components
  • Complex: Ternary complex of SpCas9-sgRNA bound to a 18-nucleotide complementary DNA substrate in the inactive state
    • RNA: sgRNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • DNA: Target strand of 18-nucleotide complementary DNA substrate
    • DNA: Non-target strand of 18-nucleotide complementary DNA substrate
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM ION
  • Ligand: water

-
Supramolecule #1: Ternary complex of SpCas9-sgRNA bound to a 18-nucleotide compleme...

SupramoleculeName: Ternary complex of SpCas9-sgRNA bound to a 18-nucleotide complementary DNA substrate in the inactive state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Streptococcus pyogenes (bacteria)

-
Macromolecule #1: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 33.005641 KDa
SequenceString:
GGGACGCAUA AAGAUGAGAC GCGUUUUAGA GCUAGAAAUA GCAAGUUAAA AUAAGGCUAG UCCGUUAUCA ACUUGAAAAA GUGGCACCG AGUCGGUGCU UUU

-
Macromolecule #2: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 158.699844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF ...String:
MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA KAILSARLSK SRRLENLIAQ LPGEKKNGLF GN LIALSLG LTPNFKSNFD LAEDAKLQLS KDTYDDDLDN LLAQIGDQYA DLFLAAKNLS DAILLSDILR VNTEITKAPL SAS MIKRYD EHHQDLTLLK ALVRQQLPEK YKEIFFDQSK NGYAGYIDGG ASQEEFYKFI KPILEKMDGT EELLVKLNRE DLLR KQRTF DNGSIPHQIH LGELHAILRR QEDFYPFLKD NREKIEKILT FRIPYYVGPL ARGNSRFAWM TRKSEETITP WNFEE VVDK GASAQSFIER MTNFDKNLPN EKVLPKHSLL YEYFTVYNEL TKVKYVTEGM RKPAFLSGEQ KKAIVDLLFK TNRKVT VKQ LKEDYFKKIE CFDSVEISGV EDRFNASLGT YHDLLKIIKD KDFLDNEENE DILEDIVLTL TLFEDREMIE ERLKTYA HL FDDKVMKQLK RRRYTGWGRL SRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSL H EHIANLAGSP AIKKGILQTV KVVDELVKVM GRHKPENIVI EMARENQTTQ KGQKNSRERM KRIEEGIKEL GSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNA KLITQRKFDN LTKAERGGLS ELDKAGFIKR QLVETRQITK HVAQILDSRM NTKYDENDKL IREVKVITLK S KLVSDFRK DFQFYKVREI NNYHHAHDAY LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YS NIMNFFK TEITLANGEI RKRPLIETNG ETGEIVWDKG RDFATVRKVL SMPQVNIVKK TEVQTGGFSK ESILPKRNSD KLI ARKKDW DPKKYGGFDS PTVAYSVLVV AKVEKGKSKK LKSVKELLGI TIMERSSFEK NPIDFLEAKG YKEVKKDLII KLPK YSLFE LENGRKRMLA SAGELQKGNE LALPSKYVNF LYLASHYEKL KGSPEDNEQK QLFVEQHKHY LDEIIEQISE FSKRV ILAD ANLDKVLSAY NKHRDKPIRE QAENIIHLFT LTNLGAPAAF KYFDTTIDRK RYTSTKEVLD ATLIHQSITG LYETRI DLS QLGGD

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

-
Macromolecule #3: Target strand of 18-nucleotide complementary DNA substrate

MacromoleculeName: Target strand of 18-nucleotide complementary DNA substrate
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.643458 KDa
SequenceString:
(DC)(DG)(DT)(DG)(DA)(DT)(DT)(DC)(DC)(DA) (DG)(DC)(DG)(DT)(DC)(DT)(DC)(DA)(DT)(DC) (DT)(DT)(DT)(DA)(DT)(DG)(DC)(DG)(DA) (DG)(DT)(DG)(DT)(DA)(DC)(DT)(DC)(DG)

-
Macromolecule #4: Non-target strand of 18-nucleotide complementary DNA substrate

MacromoleculeName: Non-target strand of 18-nucleotide complementary DNA substrate
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.73455 KDa
SequenceString:
(DC)(DG)(DA)(DG)(DT)(DA)(DC)(DA)(DC)(DT) (DA)(DT)(DT)(DG)(DC)(DG)(DG)(DG)(DA)(DC) (DG)(DC)(DT)(DA)(DT)(DT)(DA)(DT)(DT) (DG)(DG)(DA)(DA)(DT)(DC)(DA)(DC)(DG)

-
Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

-
Macromolecule #6: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

-
Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 293.15 K

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

+
Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5fq5

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 66518
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

+
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