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- EMDB-37499: Cryo-EM structure of CRISPR-Csm effector complex from Mycobacteri... -

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Basic information

Entry
Database: EMDB / ID: EMD-37499
TitleCryo-EM structure of CRISPR-Csm effector complex from Mycobacterium canettii
Map data
Sample
  • Complex: Csm effector complex
    • Protein or peptide: CRISPR system Cms protein Csm4
    • RNA: RNA (50-MER)
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • Protein or peptide: CRISPR system Cms protein Csm5
    • Protein or peptide: CRISPR system Cms protein Csm2
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
KeywordsCsm effector complex / Mycobacterium canettii / Cryo-EM structure / Csm1 / Csm2 / Csm3 / Csm4 / Csm5 / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology: / : / : / : / :
Function and homology information
Biological speciesMycobacterium canettii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsHuo Y / Ma X / Jiang T
Funding support1 items
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, 2023-
Header (metadata) releaseJul 31, 2024-
Map releaseJul 31, 2024-
UpdateJul 31, 2024-
Current statusJul 31, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37499.png

Downloads & links

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Map

FileDownload / File: emd_37499.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 384 pix.
= 399.36 Å		1.04 Å/pix.
x 384 pix.
= 399.36 Å		1.04 Å/pix.
x 384 pix.
= 399.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.004
Minimum - Maximum-0.022574814 - 0.054853544
Average (Standard dev.)-0.000013726374 (±0.0015719461)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 399.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_37499_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_37499_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Csm effector complex

EntireName: Csm effector complex
Components
  • Complex: Csm effector complex
    • Protein or peptide: CRISPR system Cms protein Csm4
    • RNA: RNA (50-MER)
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • Protein or peptide: CRISPR system Cms protein Csm5
    • Protein or peptide: CRISPR system Cms protein Csm2
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3

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Supramolecule #1: Csm effector complex

SupramoleculeName: Csm effector complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Mycobacterium canettii (bacteria)

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Macromolecule #1: CRISPR system Cms protein Csm4

MacromoleculeName: CRISPR system Cms protein Csm4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium canettii (bacteria)
Molecular weightTheoretical: 32.26477 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNSRLFRFDV VRTHFGDHGL ESSTIGCPAD TLYSALCVEA LRMGGQQLLD ELVACSTLRL TDLLPYVGPD YLVPKPLKSV RSDSSSLQK KLAKKIGFLP AAQLGSFLDG TADLDDLAAR QTKIGVHAVS AKAAIHNGKK DADPYRVGYF RFEPDAGLWL L ATGSESEL ...String:
MNSRLFRFDV VRTHFGDHGL ESSTIGCPAD TLYSALCVEA LRMGGQQLLD ELVACSTLRL TDLLPYVGPD YLVPKPLKSV RSDSSSLQK KLAKKIGFLP AAQLGSFLDG TADLDDLAAR QTKIGVHAVS AKAAIHNGKK DADPYRVGYF RFEPDAGLWL L ATGSESEL GLLTRLLKGI SALGGERTSG FGAFIPTESE VPAALTPTTA AASLMTLTTS LPTDDELEQA LAGATYRLVK RS GFVASAT YAETPRRKRD IYKLAAGSVF SRPFQGGILD VSLGGNHPVY SYARPLFLAL PESAA

UniProtKB: UNIPROTKB: G0TFC1

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Macromolecule #3: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1...

MacromoleculeName: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium canettii (bacteria)
Molecular weightTheoretical: 91.957586 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MIPALIETVI GCLLHDIGKP VQRAALGYRG RHSAIGRAFV KKIWLRDGRN PSEFADEVYE PDIEVFDRRI LDAISYHHSS ALRTAAENG RLAADAPAYV AYIADNIAAG TDRRKADSDD GQGASTWDSD TPLYSVFNRF GADTANLTFA PEMLDDREPM N IPSARRIE ...String:
MIPALIETVI GCLLHDIGKP VQRAALGYRG RHSAIGRAFV KKIWLRDGRN PSEFADEVYE PDIEVFDRRI LDAISYHHSS ALRTAAENG RLAADAPAYV AYIADNIAAG TDRRKADSDD GQGASTWDSD TPLYSVFNRF GADTANLTFA PEMLDDREPM N IPSARRIE FDKYRYTEIV NKLEAVLVDL ECSDTYLASL LNVLEATLSF VPSSTDASEV VDVSLFDHLK LTGALGACIW HY LQATGQS DFKSALFDKQ DTFYNEKAFL LTTFDVSGIQ DFIYTIHSSG AAKMLRARSF YLEMLTEHLI DELLARVGLS RAN LNYSGG GHAYLLLPNT EFARNSLEEF EREANEWLLE NFATWLFIAT GSVPLAANDL MRRPNESGPQ AHDRALRYSG LYRE LSEQL SAKKLARYSA DQLRELNSSD HDGQKGDREC SVCHTVNRTI KTINDLLLCS LCQALTAASQ QIQSESRRFL LISEE SSDG LPLPFGATLT FCSESGAKQA LQQPQTRRLY AKNKFFAGES LGTGLWVGDY VAQMEFGDYV KRASGIARLG VLRLDV DNL GQAFTHGFMK QGNGKFNTIS RTAAFSRMLS LFFRQHINYV LKHPKLRPLT GDDPERSRRA TIIYSGGDDV FVVGAWD DV IEFGIELRER FHEFTQGKLT VSAGIGMFPD KYPISVMARE VGDLEGAAKS LPGKNGVALF DPEFTFRWDE LLSKVIEE K YRHIADYFRC NEERGMAFIY KLLELLDQRG DRITEARWVY FLMRMRGPTD DTTRFQQFAN RLHQWFQDPS DAKQLKTAL HLYIYRNRKE ESE

UniProtKB: UNIPROTKB: G0TFC4

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Macromolecule #4: CRISPR system Cms protein Csm5

MacromoleculeName: CRISPR system Cms protein Csm5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium canettii (bacteria)
Molecular weightTheoretical: 42.167949 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSQYLKPFEL TLRCLGPVFI GSGEKRTPKE YVASTSMVYF PDMERLYADV AAQGKSESFE EFMMNTGKAQ PDERFNEWIA ENGVKVSPK NHGGYGVKIG SIVPGRAHRG RDGQMIQEQR QLNDIHSFIK DVLGNPYVPG SSVKGMLRSI YLQSLVHQRT A QPVRVPGH ...String:
MSQYLKPFEL TLRCLGPVFI GSGEKRTPKE YVASTSMVYF PDMERLYADV AAQGKSESFE EFMMNTGKAQ PDERFNEWIA ENGVKVSPK NHGGYGVKIG SIVPGRAHRG RDGQMIQEQR QLNDIHSFIK DVLGNPYVPG SSVKGMLRSI YLQSLVHQRT A QPVRVPGH QTREHRQYGE RFERKELRKS GRPNTRPQDA VNDLFQAIRV TDSPGLRTSD LLICQKMDVN VHGKPDGLPL FR ECLAPGT SISLRVVVDT SPTARGGWPA GERFLETLSD TVAFVNKARY AEYAAKYWDD DPQFGPIVYL GGGAGYRSKT FVT QQDDMA KVLDAQFPKI KHVAKTRDLG VSPLVLKLTK IGDKYYEMGQ CELSIRRAE

UniProtKB: UNIPROTKB: G0TFC0

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Macromolecule #5: CRISPR system Cms protein Csm2

MacromoleculeName: CRISPR system Cms protein Csm2 / type: protein_or_peptide / ID: 5 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium canettii (bacteria)
Molecular weightTheoretical: 15.006156 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSVIQDDYVK QAEQVIRGLP KKNGDFELTT TQLRVLLSLT AQLFDEAQLS SDQNLSPALR DKVQYLRVRF VYQAGREKAV RVFVERAGL LDELAQIGDS RDRLLKFCHY MEALVAYKKF LDPKETSKET E

UniProtKB: UNIPROTKB: G0TFC3

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Macromolecule #6: CRISPR system Cms endoribonuclease Csm3

MacromoleculeName: CRISPR system Cms endoribonuclease Csm3 / type: protein_or_peptide / ID: 6 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium canettii (bacteria)
Molecular weightTheoretical: 25.952537 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MITGYAKIEI TGTITVVTGL HIGAGDGFSA IGAVDKPVVR DPLTKWPMIP GTSLKGKVRT LLSRQYGADT ETFYRKPNDD PAQIRRLFG DTKEYKTGRL VFRDTKLTNK DDLEARGAKT LTEVKFENAI NRVTAKANPR QMERVIPGSE FAFSLVYEVS F GTPDEEQK ...String:
MITGYAKIEI TGTITVVTGL HIGAGDGFSA IGAVDKPVVR DPLTKWPMIP GTSLKGKVRT LLSRQYGADT ETFYRKPNDD PAQIRRLFG DTKEYKTGRL VFRDTKLTNK DDLEARGAKT LTEVKFENAI NRVTAKANPR QMERVIPGSE FAFSLVYEVS F GTPDEEQK ASLPSSDEII EDFNAIARGL KLLELDYLGG SGTRGYGQVK FSDLKARAAV GTLDRSLLEM LNHELAAV

UniProtKB: UNIPROTKB: G0TFC2

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Macromolecule #2: RNA (50-MER)

MacromoleculeName: RNA (50-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Mycobacterium canettii (bacteria)
Molecular weightTheoretical: 16.001554 KDa
SequenceString:
ACGGAAACUU AAAACCGUGU UGCACUGCAA CCCGGAAUUC UUGCACGUCG

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 110023
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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