[English] 日本語
Yorodumi
- PDB-7mi4: Symmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7mi4
TitleSymmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex
Components
  • CRISPR-associated endoribonuclease Cas2
  • CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
  • DNA (35-MER)
KeywordsHYDROLASE/DNA / CRISPR/Cas / Cas4 / PAM recognition / HYDROLASE-DNA complex
Function / homology
Function and homology information


5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / exonuclease activity / maintenance of CRISPR repeat elements / RNA endonuclease activity / 4 iron, 4 sulfur cluster binding / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / metal ion binding
Similarity search - Function
CRISPR-associated protein Cas4 / Dna2/Cas4, domain of unknown function DUF83 / Domain of unknown function DUF83 / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / CRISPR-associated endonuclease Cas1, N-terminal domain / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain ...CRISPR-associated protein Cas4 / Dna2/Cas4, domain of unknown function DUF83 / Domain of unknown function DUF83 / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / CRISPR-associated endonuclease Cas1, N-terminal domain / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1 / PD-(D/E)XK endonuclease-like domain superfamily
Similarity search - Domain/homology
: / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
Similarity search - Component
Biological speciesGeobacter sulfurreducens (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHu, C.Y. / Ke, A.K.
CitationJournal: Nature / Year: 2021
Title: Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas.
Authors: Chunyi Hu / Cristóbal Almendros / Ki Hyun Nam / Ana Rita Costa / Jochem N A Vink / Anna C Haagsma / Saket R Bagde / Stan J J Brouns / Ailong Ke /
Abstract: Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A ...Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM) and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality.
History
DepositionApr 16, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-23839
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
B: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
C: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
D: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
E: CRISPR-associated endoribonuclease Cas2
F: CRISPR-associated endoribonuclease Cas2
G: DNA (35-MER)
H: DNA (35-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)295,45718
Polymers294,3148
Non-polymers1,14310
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area31930 Å2
ΔGint-251 kcal/mol
Surface area84070 Å2

-
Components

#1: Protein
CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion


Mass: 62598.496 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA / Gene: cas4-cas1, GSU0057 / Plasmid: pET28bSUMO / Details (production host): kanamycin / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q74H36, Hydrolases; Acting on ester bonds, 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming)
#2: Protein CRISPR-associated endoribonuclease Cas2


Mass: 11190.176 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA / Gene: cas2, GSU0058 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q74H35, Hydrolases; Acting on ester bonds
#3: DNA chain DNA (35-MER)


Mass: 10769.917 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria)
#4: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mn
Has ligand of interestY

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: Symmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex
Type: COMPLEX / Details: Cas4 recognizes PAM / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.3 MDa / Experimental value: YES
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: with 5 mM DTT
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaCl
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: normal / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 6 seconds

-
Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 0.35 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 1200
EM imaging opticsPhase plate: VOLTA PHASE PLATE

-
Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC10particle selection
2EMAN9image acquisition
4EMANCTF correction
11cryoSPARCclassification
12RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120000 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00917692
ELECTRON MICROSCOPYf_angle_d0.95224262
ELECTRON MICROSCOPYf_dihedral_angle_d40.0722948
ELECTRON MICROSCOPYf_chiral_restr0.0542628
ELECTRON MICROSCOPYf_plane_restr0.0072928

+
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