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- PDB-6ifk: Cryo-EM structure of type III-A Csm-CTR1 complex, AMPPNP bound -

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Basic information

Entry
Database: PDB / ID: 6ifk
TitleCryo-EM structure of type III-A Csm-CTR1 complex, AMPPNP bound
Components
  • (Type III-A CRISPR-associated RAMP protein ...) x 3
  • (Type III-A CRISPR-associated protein ...) x 2
  • CTR1
  • crRNA
KeywordsRNA BINDING PROTEIN / Csm complex / Type III-A / CRISPR-Cas system
Function / homology
Function and homology information


exonuclease activity / transferase activity / endonuclease activity / defense response to virus / 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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / 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: 3.2 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 20, 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

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Structure visualization

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Assembly

Deposited unit
A: Type III-A CRISPR-associated protein Csm1
D: Type III-A CRISPR-associated protein Csm2
C: Type III-A CRISPR-associated protein Csm2
G: Type III-A CRISPR-associated RAMP protein Csm3
F: Type III-A CRISPR-associated RAMP protein Csm3
E: Type III-A CRISPR-associated RAMP protein Csm3
B: Type III-A CRISPR-associated RAMP protein Csm4
H: Type III-A CRISPR-associated RAMP protein Csm5
N: crRNA
J: CTR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)290,96715
Polymers289,84010
Non-polymers1,1265
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area48010 Å2
ΔGint-284 kcal/mol
Surface area84590 Å2

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Components

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

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


Mass: 86976.734 Da / Num. of mol.: 1 / Mutation: D16N
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 GFEBH

#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 NJ

#6: RNA chain crRNA


Mass: 10780.438 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus ND03 (bacteria)
#7: RNA chain CTR1


Mass: 13701.244 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus thermophilus ND03 (bacteria)

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Non-polymers , 3 types, 5 molecules

#8: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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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: Csm-CTR1 complex, AMPPNP bound / Type: COMPLEX / Details: type III-A CRISPR-Cas interference complex / Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightValue: 0.29 MDa / Experimental value: NO
Source (natural)Organism: Streptococcus thermophilus ND03 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
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 FIELD / 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
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61538 / Symmetry type: POINT

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