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- PDB-9g9k: CryoEM structure of Enterococcus italicus Csm-crRNA-CTR2 complex ... -

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Basic information

Entry
Database: PDB / ID: 9g9k
TitleCryoEM structure of Enterococcus italicus Csm-crRNA-CTR2 complex (4.3) bound to AMPNPP
Components
  • (CRISPR system ...) x 5
  • CTR
  • 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
Chem-ZAN / 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)
Severe acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.34 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
C: CRISPR system Cms protein Csm2
B: 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
R: crRNA
T: CTR
J: CRISPR system Cms protein Csm2
H: CRISPR system Cms protein Csm5
I: CRISPR system Cms endoribonuclease Csm3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)340,28216
Polymers339,22112
Non-polymers1,0614
Water00
1


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

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Components

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CRISPR system ... , 5 types, 10 molecules ACBJDEFIGH

#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.: 3
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.: 4
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
#7: 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

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RNA chain , 2 types, 2 molecules RT

#5: 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)
#6: RNA chain CTR


Mass: 15021.911 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Severe acute respiratory syndrome coronavirus 2

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Non-polymers , 2 types, 4 molecules

#8: Chemical ChemComp-ZAN / 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]adenosine


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Ternary Csm-crRNA-CTR complex bound to AMPNPP in unproductive state with (Csm1)1(Csm2)3(Csm3)4(Csm4)1(Csm5)1 stoichiometryCOMPLEX#1-#70MULTIPLE SOURCES
2Enterococcus italicus Csm-crRNA-CTR2 complex (4.3)COMPLEX#1-#5, #71RECOMBINANT
3COVID19 RNACOMPLEX#61RECOMBINANT
Molecular weightValue: 315 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Enterococcus italicus DSM 15952 (bacteria)888064
33Severe acute respiratory syndrome coronavirus 22697049
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33synthetic construct (others)32630
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
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.14 K

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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)

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56312 / Symmetry type: POINT
RefinementCross valid method: NONE

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