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- PDB-6ifu: Cryo-EM structure of type III-A Csm-CTR2-dsDNA complex -

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Basic information

Entry
Database: PDB / ID: 6ifu
TitleCryo-EM structure of type III-A Csm-CTR2-dsDNA complex
Components
  • (Type III-A CRISPR-associated RAMP protein ...) x 3
  • (Type III-A CRISPR-associated protein ...) x 2
  • CTR2
  • crRNA
KeywordsRNA BINDING PROTEIN / Csm complex / Type III-A / CRISPR-Cas system
Function / homology
Function and homology information


exonuclease activity / transferase activity / defense response to virus / endonuclease activity / RNA binding / ATP binding
Similarity search - Function
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 ...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
Similarity search - Domain/homology
RNA / RNA (> 10) / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm5 / CRISPR system Cms protein Csm2 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
Similarity search - Component
Biological speciesStreptococcus thermophilus ND03 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsYou, L. / Ma, J. / Wang, J. / Zhang, X. / Wang, Y.
CitationJournal: Cell / Year: 2019
Title: Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference.
Authors: Lilan You / Jun Ma / Jiuyu Wang / Daria Artamonova / Min Wang / Liang Liu / Hua Xiang / Konstantin Severinov / Xinzheng Zhang / Yanli Wang /
Abstract: Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, ...Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.
History
DepositionSep 21, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 12, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 19, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.year
Revision 1.2Jan 23, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3May 22, 2019Group: Data collection / Refinement description / Category: refine
Revision 1.4Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / refine
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

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Assembly

Deposited unit
A: Type III-A CRISPR-associated protein Csm1
B: Type III-A CRISPR-associated protein Csm2
C: Type III-A CRISPR-associated protein Csm2
D: Type III-A CRISPR-associated RAMP protein Csm3
E: Type III-A CRISPR-associated RAMP protein Csm3
F: Type III-A CRISPR-associated RAMP protein Csm3
G: Type III-A CRISPR-associated RAMP protein Csm4
H: Type III-A CRISPR-associated RAMP protein Csm5
I: crRNA
J: CTR2


Theoretical massNumber of molelcules
Total (without water)292,96010
Polymers292,96010
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area43590 Å2
ΔGint-250 kcal/mol
Surface area82200 Å2

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Components

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Type III-A CRISPR-associated protein ... , 2 types, 3 molecules ABC

#1: Protein Type III-A CRISPR-associated protein Csm1


Mass: 86910.648 Da / Num. of mol.: 1 / Mutation: H15A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M0F3
#2: Protein Type III-A CRISPR-associated protein Csm2


Mass: 14848.145 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Gene: csm2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M049

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Type III-A CRISPR-associated RAMP protein ... , 3 types, 5 molecules DEFGH

#3: Protein Type III-A CRISPR-associated RAMP protein Csm3


Mass: 24584.855 Da / Num. of mol.: 3 / Mutation: D33N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Gene: csm3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M035
#4: Protein Type III-A CRISPR-associated RAMP protein Csm4


Mass: 33828.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Gene: csm4 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M037
#5: Protein Type III-A CRISPR-associated RAMP protein Csm5


Mass: 41102.113 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus ND03 (bacteria)
Gene: csm5 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A2U2M038

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RNA chain , 2 types, 2 molecules IJ

#6: RNA chain crRNA


Mass: 11392.770 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus ND03 (bacteria)
#7: RNA chain CTR2


Mass: 16274.729 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus ND03 (bacteria)

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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
IDNameTypeDetailsEntity IDParent-IDSource
1type III-A Csm-CTR2-dsDNA complexCOMPLEXtype III-A Csm-CTR2-dsDNA complexall0RECOMBINANT
2type III-A Csm-CTR2-dsDNA complex, Csm1 subunitCOMPLEXtype III-A Csm-CTR2-dsDNA complex, Csm1 subunit#11RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.30 MDaYES
210.30 MDaYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Streptococcus thermophilus ND03 (bacteria)767463
32Streptococcus thermophilus ND03 (bacteria)767463
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli BL21(DE3) (bacteria)469008
32Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 60 % / Chamber temperature: 277 K

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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: BRIGHT FIELDBright-field microscopy / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 684150
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75887 / Symmetry type: POINT
RefinementResolution: 3.05→3.05 Å / Cor.coef. Fo:Fc: 0.899 / SU B: 15.559 / SU ML: 0.218 / ESU R: 0.817
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.23212 --
obs0.23212 95557 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 82.372 Å2
Baniso -1Baniso -2Baniso -3
1--0.04 Å2-0.62 Å20.57 Å2
2---0.14 Å20.75 Å2
3---0.18 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0430.01919433
ELECTRON MICROSCOPYr_bond_other_d0.0040.0217353
ELECTRON MICROSCOPYr_angle_refined_deg4.4541.90326508
ELECTRON MICROSCOPYr_angle_other_deg2.143340186
ELECTRON MICROSCOPYr_dihedral_angle_1_deg17.63552208
ELECTRON MICROSCOPYr_dihedral_angle_2_deg40.82723.993854
ELECTRON MICROSCOPYr_dihedral_angle_3_deg26.527153157
ELECTRON MICROSCOPYr_dihedral_angle_4_deg23.31815115
ELECTRON MICROSCOPYr_chiral_restr0.2570.22936
ELECTRON MICROSCOPYr_gen_planes_refined0.0190.0220714
ELECTRON MICROSCOPYr_gen_planes_other0.0040.024191
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it13.7757.618877
ELECTRON MICROSCOPYr_mcbond_other13.7647.618876
ELECTRON MICROSCOPYr_mcangle_it20.66511.33911070
ELECTRON MICROSCOPYr_mcangle_other20.66511.33911071
ELECTRON MICROSCOPYr_scbond_it17.5158.67710556
ELECTRON MICROSCOPYr_scbond_other17.5148.67810555
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other25.51112.30115438
ELECTRON MICROSCOPYr_long_range_B_refined30.27287.78624823
ELECTRON MICROSCOPYr_long_range_B_other30.27287.78424824
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.6→3.693 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.345 6968 -
Rfree-0 -
obs--100 %

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