[English] 日本語
Yorodumi
- PDB-9g9j: CryoEM structure of Enterococcus italicus Csm-crRNA complex bound... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 9g9j
TitleCryoEM structure of Enterococcus italicus Csm-crRNA complex bound to pNppA3 and AMPNPP
Components
  • (CRISPR system ...) x 5
  • crRNA
KeywordsRNA BINDING PROTEIN / Type III-A CRISPR-Cas RNA-guided nuclease Endoribonuclease cyclic oligoadenylate synthetase HD nuclease
Function / homology
Function and homology information


exonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding / ATP binding
Similarity search - Function
: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / : / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 ...: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / : / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / : / Cas10/Cmr2, second palm domain / : / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
: / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / : / RNA / RNA (> 10) / CRISPR system Cms protein Csm5 / CRISPR system Cms protein Csm4 / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm2 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
Similarity search - Component
Biological speciesEnterococcus italicus DSM 15952 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsJungfer, K. / Jinek, M.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)820152European Union
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes.
Authors: Kenny Jungfer / Štefan Moravčík / Carmela Garcia-Doval / Anna Knörlein / Jonathan Hall / Martin Jinek /
Abstract: Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign ...Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign genetic elements, which triggers the generation of cyclic oligoadenylate (cOA) second messengers by the Cas10 subunit of the type III effector complex. In turn, cOAs bind and activate ancillary effector proteins to reinforce the host immune response. Type III systems utilize distinct cOAs, including cyclic tri- (cA3), tetra- (cA4) and hexa-adenylates (cA6). However, the molecular mechanisms dictating cOA product identity are poorly understood. Here we used cryoelectron microscopy to visualize the mechanism of cA6 biosynthesis by the Csm effector complex from Enterococcus italicus (EiCsm). We show that EiCsm synthesizes oligoadenylate nucleotides in 3'-5' direction using a set of conserved binding sites in the Cas10 Palm domains to determine the size of the nascent oligoadenylate chain. Our data also reveal that conformational dynamics induced by target RNA binding results in allosteric activation of Cas10 to trigger oligoadenylate synthesis. Mutations of a key structural element in Cas10 perturb cOA synthesis to favor cA3 and cA4 formation. Together, these results provide comprehensive insights into the dynamics of cOA synthesis in type III CRISPR-Cas systems and reveal key determinants of second messenger product selectivity, thereby illuminating potential avenues for their engineering.
History
DepositionJul 25, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 29, 2025Provider: repository / Type: Initial release

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
B: CRISPR system Cms protein Csm2
C: CRISPR system Cms protein Csm2
D: CRISPR system Cms endoribonuclease Csm3
E: CRISPR system Cms endoribonuclease Csm3
F: CRISPR system Cms endoribonuclease Csm3
G: CRISPR system Cms protein Csm4
H: CRISPR system Cms protein Csm5
R: crRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)285,95613
Polymers284,1759
Non-polymers1,7814
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

-
Components

-
CRISPR system ... , 5 types, 8 molecules ABCDEFGH

#1: Protein CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / ssDNase Cas10 / Cyclic oligoadenylate synthase / EiCas10


Mass: 88294.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-term. 3C site & 10xHis tag
Source: (gene. exp.) Enterococcus italicus DSM 15952 (bacteria)
Gene: cas10, csm1, HMPREF9088_1947 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: E6LHV7, Hydrolases; Acting on ester bonds, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: Protein CRISPR system Cms protein Csm2 / CRISPR type III A-associated protein Csm2


Mass: 16523.047 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterococcus italicus DSM 15952 (bacteria)
Gene: csm2, HMPREF9088_1946 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: E6LHV6
#3: Protein CRISPR system Cms endoribonuclease Csm3 / CRISPR type III A-associated RAMP protein Csm3


Mass: 23500.777 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: E32A
Source: (gene. exp.) Enterococcus italicus DSM 15952 (bacteria)
Gene: csm3, HMPREF9088_1945 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: E6LHV5, Hydrolases; Acting on ester bonds
#4: Protein CRISPR system Cms protein Csm4 / CRISPR type III A-associated RAMP protein Csm4


Mass: 34358.059 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterococcus italicus DSM 15952 (bacteria)
Gene: csm4, HMPREF9088_1944 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: E6LHV4
#5: Protein CRISPR system Cms protein Csm5 / CRISPR type III A-associated protein Csm5


Mass: 43328.676 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-term. 3C site & Twin-Strep tag
Source: (gene. exp.) Enterococcus italicus DSM 15952 (bacteria)
Gene: csm5, HMPREF9088_1943 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: E6LHV3

-
RNA chain , 1 types, 1 molecules R

#6: RNA chain crRNA


Mass: 14645.835 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterococcus italicus DSM 15952 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)

-
Non-polymers , 3 types, 4 molecules

#7: Chemical ChemComp-A1II9 / adenosine-5'-[(beta,gamma)-imido]triphosphate-adenosine-monophosphate-adenosine-monophosphate / [[[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3-[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3-[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl]oxy-4-oxidanyl-oxolan-2-yl]methoxy-oxidanyl-phosphoryl]oxy-4-oxidanyl-oxolan-2-yl]methoxy-oxidanyl-phosphoryl]oxy-oxidanyl-phosphoryl]amino]phosphonic acid / pNppA3


Mass: 1164.608 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C30H41N16O24P5 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#9: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION

-
Details

Has ligand of interestY
Has protein modificationY

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: Binary Csm-crRNA complex bound to pNppA3 and AMPPNP with (Csm1)1(Csm2)2(Csm3)3(Csm4)1(Csm5)1 stoichiometry
Type: COMPLEX / Entity ID: #1-#4, #6, #5 / Source: RECOMBINANT
Molecular weightValue: 0.26 MDa / Experimental value: NO
Source (natural)Organism: Enterococcus italicus DSM 15952 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
2350 mMpotassium chlorideKCl1
310 mMmagnesium chlorideMgCl1
41 mMdithiothreitolDTT1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.14 K

-
Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 66.51 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

-
Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53176 / Symmetry type: POINT
RefinementCross valid method: NONE

+
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