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- PDB-8x5d: The cryo-EM structure of the Mycobacterium tuberculosis CRISPR-Cs... -

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Basic information

Entry
Database: PDB / ID: 8x5d
TitleThe cryo-EM structure of the Mycobacterium tuberculosis CRISPR-Csm complex
Components
  • CRISPR system Cms endoribonuclease Csm3
  • CRISPR system Cms protein Csm5
  • Csm2
  • RNA (47-MER)
KeywordsRNA BINDING PROTEIN / Mycobacteria CRISPR-Csm complexes
Function / homology
Function and homology information


endonuclease activity / defense response to virus / RNA binding
Similarity search - Function
CRISPR-associated protein Csm5 / CRISPR-associated RAMP Csm3 / CRISPR type III-associated protein / RAMP superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm5
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLiu, M.X. / Li, Z.K.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82225028 China
Citation
Journal: To Be Published
Title: The cryo-EM structure of the Mycobacterium tuberculosis CRISPR-Csm complex
Authors: Liu, M.X. / Li, Z.K.
#1: Journal: FASEB J / Year: 2019
Title: Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.
Authors: Wenjing Wei / Shuai Zhang / Joy Fleming / Ying Chen / Zihui Li / Shanghua Fan / Yi Liu / Wei Wang / Ting Wang / Ying Liu / Baiguang Ren / Ming Wang / Jianjian Jiao / Yuanyuan Chen / Ying ...Authors: Wenjing Wei / Shuai Zhang / Joy Fleming / Ying Chen / Zihui Li / Shanghua Fan / Yi Liu / Wei Wang / Ting Wang / Ying Liu / Baiguang Ren / Ming Wang / Jianjian Jiao / Yuanyuan Chen / Ying Zhou / Yafeng Zhou / Shoujin Gu / Xiaoli Zhang / Li Wan / Tao Chen / Lin Zhou / Yong Chen / Xian-En Zhang / Chuanyou Li / Hongtai Zhang / Lijun Bi /
Abstract: Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus ...Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis, the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III-A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA-seq approaches, we show it is active in invader defense and has features atypical of type III-A systems: mature CRISPR RNA (crRNA) in its crRNA-CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3'-end processing after Cas6 cleavage of repeat RNA 8 nt from its 3' end. crRNAs generated resemble mature crRNA in type I systems, having both 5' (8 nt) and 3' (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 3' hairpin in the repeat RNA and the sequence of its stem base nucleotides. This study unveils further diversity among CRISPR/Cas systems and provides insight into the crRNA recognition mechanism in M. tuberculosis, providing a foundation for investigating the potential of a type III-A-based genome editing system.-Wei, W., Zhang, S., Fleming, J., Chen, Y., Li, Z., Fan, S., Liu, Y., Wang, W., Wang, T., Liu, Y., Ren, B., Wang, M., Jiao, J., Chen, Y., Zhou, Y., Zhou, Y., Gu, S., Zhang, X., Wan, L., Chen, T., Zhou, L., Chen, Y., Zhang, X.-E., Li, C., Zhang, H., Bi, L. Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.
History
DepositionNov 17, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
K: CRISPR system Cms endoribonuclease Csm3
G: CRISPR system Cms endoribonuclease Csm3
N: CRISPR system Cms protein Csm5
L: CRISPR system Cms endoribonuclease Csm3
O: RNA (47-MER)
J: CRISPR system Cms endoribonuclease Csm3
I: CRISPR system Cms endoribonuclease Csm3
H: CRISPR system Cms endoribonuclease Csm3
F: Csm2
E: Csm2
D: Csm2
C: Csm2
B: Csm2


Theoretical massNumber of molelcules
Total (without water)335,73213
Polymers335,73213
Non-polymers00
Water0
1


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

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Components

#1: Protein
CRISPR system Cms endoribonuclease Csm3


Mass: 26147.736 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: CAB90_03150 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A045JG98
#2: Protein CRISPR system Cms protein Csm5


Mass: 42752.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: csm5 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0T5YG06
#3: RNA chain RNA (47-MER)


Mass: 59360.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Production host: Escherichia coli (E. coli)
#4: Protein
Csm2


Mass: 15346.579 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Production host: Escherichia coli (E. coli)

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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: Mycobacteria CRISPR-Csm complexes / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (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 / 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 / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 119066 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00318863
ELECTRON MICROSCOPYf_angle_d0.61925652
ELECTRON MICROSCOPYf_dihedral_angle_d14.80111440
ELECTRON MICROSCOPYf_chiral_restr0.0442946
ELECTRON MICROSCOPYf_plane_restr0.0063158

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