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- PDB-6nue: Small conformation of apo CRISPR_Csm complex -

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Basic information

Entry
Database: PDB / ID: 6nue
TitleSmall conformation of apo CRISPR_Csm complex
Components
  • (CRISPR system ...) x 2
  • (CRISPR type III-associated RAMP protein ...) x 2
  • crRNA
KeywordsHYDROLASE / TRANSFERASE/RNA / CRISPR / Type III-A / ssRNAase / ssDNase / TRANSFERASE-RNA complex
Function / homology
Function and homology information


exonuclease activity / defense response to virus / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding / ATP binding
Csm1 subunit domain B / HD domain / HD domain / CRISPR Csm4 C-terminal domain / RAMP superfamily / Csm2 Type III-A / Csm1, subunit domain B / Csm4, C-terminal / CRISPR-associated RAMP Csm3 / CRISPR-associated protein, Csm2 Type III-A ...Csm1 subunit domain B / HD domain / HD domain / CRISPR Csm4 C-terminal domain / RAMP superfamily / Csm2 Type III-A / Csm1, subunit domain B / Csm4, C-terminal / CRISPR-associated RAMP Csm3 / CRISPR-associated protein, Csm2 Type III-A / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / CRISPR type III-associated protein / CRISPR type III-associated RAMP protein Csm4 / GGDEF domain
CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm4 / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm2
Biological speciesStreptococcus thermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsZhang, K. / Pintilie, G. / Li, S. / Zhu, Y. / Chiu, W. / Huang, Z.
Funding support United States, China, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)P41GM103832 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)U54GM103297 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R01GM079429 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10OD021600 United States
National Natural Science Foundation of China (NSFC)31825008 China
National Natural Science Foundation of China (NSFC)31422014 China
CitationJournal: Cell Res. / Year: 2019
Title: Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry.
Authors: Minghui Guo / Kaiming Zhang / Yuwei Zhu / Grigore D Pintilie / Xiaoyu Guan / Shanshan Li / Michael F Schmid / Zhuo Ma / Wah Chiu / Zhiwei Huang /
Abstract: The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading ...The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown. Here we report cryo-EM structures of the Csm-crRNA complex, with or without target ssRNA, at near-atomic resolution. Our cryo-EM maps allow us to build atomic models of the key macromolecular components, including Cas10, Csm2, Csm3, Csm4, crRNA and the invading ssRNA. Our structure resolves unambiguously the stoichiometry and tertiary structures of the Csm protein complex and the interactions between protein components and the crRNA/ssRNA. Interestingly, the new atomic structures of the Csm proteins presented here are similar to those of previously known Csm proteins in other species despite their low sequence similarity. Our combined structural and biochemical data suggest that ssRNA cleavage is preferentially carried out near its 5'-end, that the extent of interactions among the ssRNA, crRNA and the protein components regulates the DNase activity of the Csm complex, and that the 3' flanking sequence of ssRNA activates the Cas10 DNase activity allosterically.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJan 31, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
J: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
A: CRISPR system Cms protein Csm2
B: CRISPR system Cms protein Csm2
C: CRISPR type III-associated RAMP protein Csm3
E: CRISPR type III-associated RAMP protein Csm3
H: crRNA
I: CRISPR type III-associated RAMP protein Csm4
M: CRISPR system Cms protein Csm2
N: CRISPR type III-associated RAMP protein Csm3
O: CRISPR type III-associated RAMP protein Csm3
P: CRISPR type III-associated RAMP protein Csm3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)309,88012
Polymers309,37311
Non-polymers5071
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area45130 Å2
ΔGint-238 kcal/mol
Surface area108490 Å2

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Components

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CRISPR system ... , 2 types, 4 molecules JABM

#1: Protein CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / ssDNase Cas10 / Cyclic oligoadenylate synthase / StCas10


Mass: 86930.672 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: cas10, csm1
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0A7HFE1, 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: 14238.391 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csm2
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0A7HIX1

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CRISPR type III-associated RAMP protein ... , 2 types, 6 molecules CENOPI

#3: Protein
CRISPR type III-associated RAMP protein Csm3 / Csm3 protein


Mass: 24600.918 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csm3, CDA68_00842
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0A7HIF0
#5: Protein CRISPR type III-associated RAMP protein Csm4 / Csm4 protein


Mass: 33786.949 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csm4, CDA68_00841
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0A7HGA1

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RNA chain / Non-polymers , 2 types, 2 molecules H

#4: RNA chain crRNA


Mass: 22935.553 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#6: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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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: Small conformation of apo CRISPR_Csm complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.6
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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
Image recordingElectron dose: 7 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
7UCSF Chimeramodel fitting
9PHENIX1.14model refinement
12cryoSPARC0.65classification
13cryoSPARC0.653D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81540 / Symmetry type: POINT

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