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- PDB-9ash: Cryo-EM structure of the active Lactococcus lactis Csm bound to t... -

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Basic information

Entry
Database: PDB / ID: 9ash
TitleCryo-EM structure of the active Lactococcus lactis Csm bound to target in post-cleavage stage
Components
  • (CRISPR system ...) x 4
  • CRISPR RNA
  • CRISPR-associated protein Csm4
  • RNA (5'-D(*(ATP))-R(P*AP*A)-3')
  • Target RNA
KeywordsRNA BINDING PROTEIN/RNA / Type III-A CRISPR-Cas / Csm / Cyclic Oligoadenylate synthesis / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


exonuclease activity / endonuclease activity / defense response to virus / 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 / : / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / HD domain / HD domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / RNA / RNA (> 10) / CRISPR system Cms endoribonuclease Csm3 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm2 / CRISPR system Cms protein Csm5
Similarity search - Component
Biological speciesLactococcus lactis subsp. lactis (lactic acid bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.58 Å
AuthorsWang, B. / Goswami, H.N. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM099604 United States
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Molecular basis for cA6 synthesis by a type III-A CRISPR-Cas enzyme and its conversion to cA4 production.
Authors: Hemant N Goswami / Fozieh Ahmadizadeh / Bing Wang / Doreen Addo-Yobo / Yu Zhao / A Carl Whittington / Huan He / Michael P Terns / Hong Li /
Abstract: The type III-A (Csm) CRISPR-Cas systems are multi-subunit and multipronged prokaryotic enzymes in guarding the hosts against viral invaders. Beyond cleaving activator RNA transcripts, Csm confers ...The type III-A (Csm) CRISPR-Cas systems are multi-subunit and multipronged prokaryotic enzymes in guarding the hosts against viral invaders. Beyond cleaving activator RNA transcripts, Csm confers two additional activities: shredding single-stranded DNA and synthesizing cyclic oligoadenylates (cOAs) by the Cas10 subunit. Known Cas10 enzymes exhibit a fascinating diversity in cOA production. Three major forms-cA3, cA4 and cA6have been identified, each with the potential to trigger unique downstream effects. Whereas the mechanism for cOA-dependent activation is well characterized, the molecular basis for synthesizing different cOA isoforms remains unclear. Here, we present structural characterization of a cA6-producing Csm complex during its activation by an activator RNA. Analysis of the captured intermediates of cA6 synthesis suggests a 3'-to-5' nucleotidyl transferring process. Three primary adenine binding sites can be identified along the chain elongation path, including a unique tyrosine-threonine dyad found only in the cA6-producing Cas10. Consistently, disrupting the tyrosine-threonine dyad specifically impaired cA6 production while promoting cA4 production. These findings suggest that Cas10 utilizes a unique enzymatic mechanism for forming the phosphodiester bond and has evolved distinct strategies to regulate the cOA chain length.
History
DepositionFeb 25, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
B: CRISPR-associated protein Csm4
F: CRISPR system Cms endoribonuclease Csm3
H: CRISPR system Cms endoribonuclease Csm3
G: CRISPR system Cms endoribonuclease Csm3
I: CRISPR system Cms endoribonuclease Csm3
R: CRISPR RNA
D: CRISPR system Cms protein Csm2
E: CRISPR system Cms protein Csm2
T: Target RNA
C: CRISPR system Cms protein Csm2
J: CRISPR system Cms protein Csm5
P: RNA (5'-D(*(ATP))-R(P*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)333,63817
Polymers333,05813
Non-polymers5804
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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CRISPR system ... , 4 types, 9 molecules AFHGIDECJ

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


Mass: 87365.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: cas10 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CEJ3
#3: Protein
CRISPR system Cms endoribonuclease Csm3 / CRISPR type III A-associated RAMP protein Csm3


Mass: 23869.170 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: csm3 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CEA3
#5: Protein CRISPR system Cms protein Csm2 / CRISPR type III A-associated protein Csm2


Mass: 17014.330 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: csm2 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CFW2
#7: Protein CRISPR system Cms protein Csm5 / CRISPR type III A-associated protein Csm5


Mass: 40621.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: csm5 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CG31

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Protein , 1 types, 1 molecules B

#2: Protein CRISPR-associated protein Csm4


Mass: 33943.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: csm4 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CFH1

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RNA chain , 3 types, 3 molecules RTP

#4: RNA chain CRISPR RNA


Mass: 11946.271 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
#6: RNA chain Target RNA


Mass: 11558.874 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
#8: RNA chain RNA (5'-D(*(ATP))-R(P*AP*A)-3')


Mass: 1102.620 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Production host: Escherichia coli (E. coli)

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Non-polymers , 3 types, 6 molecules

#9: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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

ComponentName: ATP bound Lactococcus lactis Csm CRISPR-Cas complex with stable Cas10
Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 965374 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00322815
ELECTRON MICROSCOPYf_angle_d0.56831025
ELECTRON MICROSCOPYf_dihedral_angle_d10.8093570
ELECTRON MICROSCOPYf_chiral_restr0.0423510
ELECTRON MICROSCOPYf_plane_restr0.0033756

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