[English] 日本語
Yorodumi
- EMDB-41867: SpG Cas9 with NGG PAM DNA target -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-41867
TitleSpG Cas9 with NGG PAM DNA target
Map data
Sample
  • Complex: SpG Cas9 with NGG PAM target
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: RNA (98-MER)
    • DNA: DNA (5'-D(P*CP*GP*TP*TP*TP*GP*TP*AP*CP*TP*CP*CP*AP*GP*CP*G)-3')
    • DNA: DNA (5'-D(P*TP*CP*TP*CP*AP*TP*CP*TP*TP*TP*AP*TP*GP*CP*GP*TP*C)-3')
    • DNA: DNA (5'-D(P*TP*GP*GP*AP*GP*TP*AP*CP*AP*AP*AP*CP*G)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsSpG-Cas9 / CRISPR / Cas9 / IMMUNE SYSTEM / R-loop / IMMUNE SYSTEM-DNA-RNA 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
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsBravo JPK / Hibshman GN / Taylor DW
Funding support United States, 2 items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Nat Commun / Year: 2024
Title: Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.
Authors: Grace N Hibshman / Jack P K Bravo / Matthew M Hooper / Tyler L Dangerfield / Hongshan Zhang / Ilya J Finkelstein / Kenneth A Johnson / David W Taylor /
Abstract: CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable ...CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.
History
DepositionSep 8, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_41867.png

Downloads & links

-
Map

FileDownload / File: emd_41867.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.833 Å
Density
Contour LevelBy AUTHOR: 0.521
Minimum - Maximum-1.5552142 - 3.0123417
Average (Standard dev.)-0.00016997912 (±0.045354247)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 426.496 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: #1

Fileemd_41867_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: #2

Fileemd_41867_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : SpG Cas9 with NGG PAM target

EntireName: SpG Cas9 with NGG PAM target
Components
  • Complex: SpG Cas9 with NGG PAM target
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • RNA: RNA (98-MER)
    • DNA: DNA (5'-D(P*CP*GP*TP*TP*TP*GP*TP*AP*CP*TP*CP*CP*AP*GP*CP*G)-3')
    • DNA: DNA (5'-D(P*TP*CP*TP*CP*AP*TP*CP*TP*TP*TP*AP*TP*GP*CP*GP*TP*C)-3')
    • DNA: DNA (5'-D(P*TP*GP*GP*AP*GP*TP*AP*CP*AP*AP*AP*CP*G)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: water

-
Supramolecule #1: SpG Cas9 with NGG PAM target

SupramoleculeName: SpG Cas9 with NGG PAM target / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 211.54 KDa

-
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
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 158.476781 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL QEIFSNEMA KVDDSFFHRL EESFLVEEDK KHERHPIFGN IVDEVAYHEK YPTIYHLRKK LVDSTDKADL RLIYLALAHM I KFRGHFLI ...String:
KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL QEIFSNEMA KVDDSFFHRL EESFLVEEDK KHERHPIFGN IVDEVAYHEK YPTIYHLRKK LVDSTDKADL RLIYLALAHM I KFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN LI ALSLGLT PNFKSNFDLA EDAKLQLSKD TYDDDLDNLL AQIGDQYADL FLAAKNLSDA ILLSDILRVN TEITKAPLSA SMI KRYDEH HQDLTLLKAL VRQQLPEKYK EIFFDQSKNG YAGYIDGGAS QEEFYKFIKP ILEKMDGTEE LLVKLNREDL LRKQ RTFDN GSIPHQIHLG ELHAILRRQE DFYPFLKDNR EKIEKILTFR IPYYVGPLAR GNSRFAWMTR KSEETITPWN FEEVV DKGA SAQSFIERMT NFDKNLPNEK VLPKHSLLYE YFTVYNELTK VKYVTEGMRK PAFLSGEQKK AIVDLLFKTN RKVTVK QLK EDYFKKIECF DSVEISGVED RFNASLGTYH DLLKIIKDKD FLDNEENEDI LEDIVLTLTL FEDREMIEER LKTYAHL FD DKVMKQLKRR RYTGWGRLSR KLINGIRDKQ SGKTILDFLK SDGFANRNFM QLIHDDSLTF KEDIQKAQVS GQGDSLHE H IANLAGSPAI KKGILQTVKV VDELVKVMGR HKPENIVIEM ARENQTTQKG QKNSRERMKR IEEGIKELGS QILKEHPVE NTQLQNEKLY LYYLQNGRDM YVDQELDINR LSDYDVDHIV PQSFLKDDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY WRQLLNAKL ITQRKFDNLT KAERGGLSEL DKAGFIKRQL VETRQITKHV AQILDSRMNT KYDENDKLIR EVKVITLKSK L VSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS NI MNFFKTE ITLANGEIRK RPLIETNGET GEIVWDKGRD FATVRKVLSM PQVNIVKKTE VQTGGFSKES ILPKRNSDKL IAR KKDWDP KKYGGFLWPT VAYSVLVVAK VEKGKSKKLK SVKELLGITI MERSSFEKNP IDFLEAKGYK EVKKDLIIKL PKYS LFELE NGRKRMLASA KQLQKGNELA LPSKYVNFLY LASHYEKLKG SPEDNEQKQL FVEQHKHYLD EIIEQISEFS KRVIL ADAN LDKVLSAYNK HRDKPIREQA ENIIHLFTLT NLGAPAAFKY FDTTIDRKQY RSTKEVLDAT LIHQSITGLY ETRIDL SQL G

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

-
Macromolecule #2: RNA (98-MER)

MacromoleculeName: RNA (98-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 31.7029 KDa
SequenceString:
GACGCAUAAA GAUGAGACGC GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAG UCGGUGCUU

-
Macromolecule #3: DNA (5'-D(P*CP*GP*TP*TP*TP*GP*TP*AP*CP*TP*CP*CP*AP*GP*CP*G)-3')

MacromoleculeName: DNA (5'-D(P*CP*GP*TP*TP*TP*GP*TP*AP*CP*TP*CP*CP*AP*GP*CP*G)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.865152 KDa
SequenceString:
(DC)(DG)(DT)(DT)(DT)(DG)(DT)(DA)(DC)(DT) (DC)(DC)(DA)(DG)(DC)(DG)

-
Macromolecule #4: DNA (5'-D(P*TP*CP*TP*CP*AP*TP*CP*TP*TP*TP*AP*TP*GP*CP*GP*TP*C)-3')

MacromoleculeName: DNA (5'-D(P*TP*CP*TP*CP*AP*TP*CP*TP*TP*TP*AP*TP*GP*CP*GP*TP*C)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.119319 KDa
SequenceString:
(DT)(DC)(DT)(DC)(DA)(DT)(DC)(DT)(DT)(DT) (DA)(DT)(DG)(DC)(DG)(DT)(DC)

-
Macromolecule #5: DNA (5'-D(P*TP*GP*GP*AP*GP*TP*AP*CP*AP*AP*AP*CP*G)-3')

MacromoleculeName: DNA (5'-D(P*TP*GP*GP*AP*GP*TP*AP*CP*AP*AP*AP*CP*G)-3')
type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.024649 KDa
SequenceString:
(DT)(DG)(DG)(DA)(DG)(DT)(DA)(DC)(DA)(DA) (DA)(DC)(DG)

-
Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

-
Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 5 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 59087

+
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