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Yorodumi- PDB-8x5d: The cryo-EM structure of the Mycobacterium tuberculosis CRISPR-Cs... -
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-Basic information
Entry | Database: PDB / ID: 8x5d | ||||||
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Title | The cryo-EM structure of the Mycobacterium tuberculosis CRISPR-Csm complex | ||||||
Components |
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Keywords | RNA BINDING PROTEIN / Mycobacteria CRISPR-Csm complexes | ||||||
Function / homology | Function and homology information | ||||||
Biological species | Mycobacterium tuberculosis (bacteria) Mycobacterium canettii (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||
Authors | Liu, M.X. / Li, Z.K. | ||||||
Funding support | China, 1items
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Citation | Journal: 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. #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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8x5d.cif.gz | 410.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8x5d.ent.gz | 334.6 KB | Display | PDB format |
PDBx/mmJSON format | 8x5d.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8x5d_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 8x5d_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 8x5d_validation.xml.gz | 69.6 KB | Display | |
Data in CIF | 8x5d_validation.cif.gz | 105.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/x5/8x5d ftp://data.pdbj.org/pub/pdb/validation_reports/x5/8x5d | HTTPS FTP |
-Related structure data
Related structure data | 38066MC 8wfxC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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 | | 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 | | 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 | Mass: 15346.579 Da / Num. of mol.: 5 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mycobacterium canettii (bacteria) / Production host: Escherichia coli (E. coli) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component |
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Source (natural) |
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Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Humidity: 100 % |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 119066 / Symmetry type: POINT | ||||||||||||||||||||||||
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