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- PDB-8wfx: Cryo-EM structure of CRISPR-Csm effector complex from Mycobacteri... -

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Basic information

Entry
Database: PDB / ID: 8wfx
TitleCryo-EM structure of CRISPR-Csm effector complex from Mycobacterium canettii
Components
  • (CRISPR system ...) x 5
  • RNA (50-MER)
KeywordsRNA BINDING PROTEIN/RNA / Csm effector complex / Mycobacterium canettii / Cryo-EM structure / Csm1 / Csm2 / Csm3 / Csm4 / Csm5 / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10) / : / : / : / : / :
Function and homology information
Biological speciesMycobacterium canettii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsHuo, Y. / Ma, X. / Jiang, T.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Int J Biol Macromol / Year: 2024
Title: Type-III-A structure of mycobacteria CRISPR-Csm complexes involving atypical crRNAs.
Authors: Hongtai Zhang / Mingmin Shi / Xiaoli Ma / Mengxi Liu / Nenhan Wang / Qiuhua Lu / Zekai Li / Yanfeng Zhao / Hongshen Zhao / Hong Chen / Huizhi Zhang / Tao Jiang / Songying Ouyang / Yangao Huo / Lijun Bi /
Abstract: Tuberculosis (TB), a leading cause of mortality globally, is a chronic infectious disease caused by Mycobacterium tuberculosis that primarily infiltrates the lung. The mature crRNAs in M. ...Tuberculosis (TB), a leading cause of mortality globally, is a chronic infectious disease caused by Mycobacterium tuberculosis that primarily infiltrates the lung. The mature crRNAs in M. tuberculosis transcribed from the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus exhibit an atypical structure featured with 5' and 3' repeat tags at both ends of the intact crRNA, in contrast to typical Type-III-A crRNAs that possess 5' repeat tags and partial crRNA sequences. However, this structural peculiarity particularly concerning the specific binding characteristics of the 3' repeat end within the mature crRNA within the Csm complex, has not been comprehensively elucidated. Here, our Mycobacteria CRISPR-Csm complexes structure represents the largest Csm complex reported to date. It incorporates an atypical Type-III-A CRISPR RNA (crRNA) (46 nt) with 5' 8-nt and 3' 4-nt repeat sequences in the stoichiometry of Mycobacteria Csm12345 The PAM-independent single-stranded RNAs (ssRNAs) are the most suitable substrate for the Csm complex. The 3'-repeat end trimming of mature crRNA was not necessary for its cleavage activity in Type-III-A Csm complex. Our work broadens our understanding of the Type-III-A Csm complex and identifies another mature crRNA processing mechanism in the Type-III-A CRISPR-Cas system based on structural biology.
History
DepositionSep 20, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 31, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
M: CRISPR system Cms protein Csm4
O: RNA (50-MER)
A: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
N: CRISPR system Cms protein Csm5
E: CRISPR system Cms protein Csm2
C: CRISPR system Cms protein Csm2
B: CRISPR system Cms protein Csm2
D: CRISPR system Cms protein Csm2
F: CRISPR system Cms protein Csm2
I: CRISPR system Cms endoribonuclease Csm3
J: CRISPR system Cms endoribonuclease Csm3
K: CRISPR system Cms endoribonuclease Csm3
L: CRISPR system Cms endoribonuclease Csm3
G: CRISPR system Cms endoribonuclease Csm3
H: CRISPR system Cms endoribonuclease Csm3


Theoretical massNumber of molelcules
Total (without water)413,13815
Polymers413,13815
Non-polymers00
Water00
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 ... , 5 types, 14 molecules MANECBDFIJKLGH

#1: Protein CRISPR system Cms protein Csm4


Mass: 32264.770 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium canettii (bacteria) / Gene: MCAN_28441 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G0TFC1
#3: Protein CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)


Mass: 91957.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium canettii (bacteria) / Gene: MCAN_28471 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G0TFC4
#4: Protein CRISPR system Cms protein Csm5


Mass: 42167.949 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium canettii (bacteria) / Gene: MCAN_28431 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G0TFC0
#5: Protein
CRISPR system Cms protein Csm2


Mass: 15006.156 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium canettii (bacteria) / Gene: MCAN_28461 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G0TFC3
#6: Protein
CRISPR system Cms endoribonuclease Csm3


Mass: 25952.537 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium canettii (bacteria) / Gene: MCAN_28451 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G0TFC2

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RNA chain , 1 types, 1 molecules O

#2: RNA chain RNA (50-MER)


Mass: 16001.554 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium canettii (bacteria) / Production host: Escherichia coli BL21(DE3) (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

ComponentName: Csm effector complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium canettii (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
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: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110023 / Symmetry type: POINT

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