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- EMDB-38066: The cryo-EM structure of the Mycobacterium tuberculosis CRISPR-Cs... -

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Basic information

Entry
Database: EMDB / ID: EMD-38066
TitleThe cryo-EM structure of the Mycobacterium tuberculosis CRISPR-Csm complex
Map data
Sample
  • Complex: Mycobacteria CRISPR-Csm complexes
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • Protein or peptide: CRISPR system Cms protein Csm5
    • RNA: RNA (47-MER)
    • Protein or peptide: Csm2
KeywordsMycobacteria CRISPR-Csm complexes / RNA BINDING PROTEIN
Function / homologyCRISPR-associated protein Csm5 / CRISPR-associated RAMP Csm3 / CRISPR type III-associated protein / RAMP superfamily / endonuclease activity / defense response to virus / RNA binding / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm5
Function and homology information
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLiu MX / Li ZK
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82225028 China
CitationJournal: 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, 2023-
Header (metadata) releaseMar 6, 2024-
Map releaseMar 6, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38066.png

Downloads & links

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Map

FileDownload / File: emd_38066.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.855 Å
Density
Contour LevelBy AUTHOR: 0.349
Minimum - Maximum-1.2616274 - 3.1738565
Average (Standard dev.)-0.00023194296 (±0.04985265)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 427.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_38066_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_38066_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mycobacteria CRISPR-Csm complexes

EntireName: Mycobacteria CRISPR-Csm complexes
Components
  • Complex: Mycobacteria CRISPR-Csm complexes
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • Protein or peptide: CRISPR system Cms protein Csm5
    • RNA: RNA (47-MER)
    • Protein or peptide: Csm2

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Supramolecule #1: Mycobacteria CRISPR-Csm complexes

SupramoleculeName: Mycobacteria CRISPR-Csm complexes / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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Macromolecule #1: CRISPR system Cms endoribonuclease Csm3

MacromoleculeName: CRISPR system Cms endoribonuclease Csm3 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 26.147736 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAHMTTSYAK IEITGTLTVL TGLQIGAGDG FSAIGAVDKP VVRDPLSRLP MIPGTSLKGK VRTLLSRQYG ADTETFYRKP NEDHAHIRR LFGDTEEYMT GRLVFRDTKL TNKDDLEARG AKTLTEVKFE NAINRVTAKA NLRQMERVIP GSEFAFSLVY E VSFGTPGE ...String:
MAHMTTSYAK IEITGTLTVL TGLQIGAGDG FSAIGAVDKP VVRDPLSRLP MIPGTSLKGK VRTLLSRQYG ADTETFYRKP NEDHAHIRR LFGDTEEYMT GRLVFRDTKL TNKDDLEARG AKTLTEVKFE NAINRVTAKA NLRQMERVIP GSEFAFSLVY E VSFGTPGE EQKASLPSSD EIIEDFNAIA RGLKLLELDY LGGSGTRGYG QVKFSNLKAR AAVGALDGSL LEKLNHELAA V

UniProtKB: CRISPR system Cms endoribonuclease Csm3

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Macromolecule #2: CRISPR system Cms protein Csm5

MacromoleculeName: CRISPR system Cms protein Csm5 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 42.752129 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAHMNTYLKP FELTLRCLGP VFIGSGEKRT SKEYHVEGDR VYFPDMELLY ADIPAHKRKS FEAFVMNTDG AQATAPLKEW VEPNAVKLD PAKHRGYEVK IGSIEPRRAS RGRGGRMTRK KLTLNEIHAF IKDPLGRPYV PGSTVKGMLR SIYLQSLVHK R TAQPVRVP ...String:
MAHMNTYLKP FELTLRCLGP VFIGSGEKRT SKEYHVEGDR VYFPDMELLY ADIPAHKRKS FEAFVMNTDG AQATAPLKEW VEPNAVKLD PAKHRGYEVK IGSIEPRRAS RGRGGRMTRK KLTLNEIHAF IKDPLGRPYV PGSTVKGMLR SIYLQSLVHK R TAQPVRVP GHQTREHRQY GERFERKELR KSGRPNTRPQ DAVNDLFQAI RVTDSPALRT SDLLICQKMD MNVHGKPDGL PL FRECLAP GTSISHRVVV DTSPTARGGW REGERFLETL AETAASVNQA RYAEYRAMYP GVNAIVGPIV YLGGGAGYRS KTF VTDQDD MAKVLDAQFG KVVKHVDKTR ELRVSPLVLK RTKIDNICYE MGQCELSIRR AE

UniProtKB: CRISPR system Cms protein Csm5

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Macromolecule #4: Csm2

MacromoleculeName: Csm2 / type: protein_or_peptide / ID: 4 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 15.346579 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAHMSVIQDD YVKQAEQVIR GLPKKNGDFE LTTTQLRVLL SLTAQLFDEA QLSSDQNLSP ALRDKVQYLR VRFVYQAGRE KAVRVFVER AGLLDELAQI GDSRDRLLKF CHYMEALVAY KKFLDPKETS KETE

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Macromolecule #3: RNA (47-MER)

MacromoleculeName: RNA (47-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 59.360578 KDa
SequenceString:
GUCGUCAGAC CCAAAACCCC GAGAGGGGAC GGAAACUUAA AACCGUGUUG CACUGCAACC CGGAAUUCUU GCACGUCGUC AGACCCAAA ACCCCGAGAG GGGACGGAAA CUUAAAACCG UGUUGCACUG CAACCCGGAA UUCUUGCACG UCGUCAGACC C AAAACCCC GAGAGGGGAC GGAAAC

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 119066

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