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- EMDB-22267: Structure of the Lactococcus lactis Csm CTR_3:2 CRISPR-Cas Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-22267
TitleStructure of the Lactococcus lactis Csm CTR_3:2 CRISPR-Cas Complex
Map data
Sample
  • Complex: Lactococcus lactis Csm CRISPR-Cas CTR_3:2 Complex
    • Protein or peptide: CRISPR-associated protein Csm4
    • Protein or peptide: CRISPR-associated protein Csm3
    • RNA: Crispr RNA
    • Protein or peptide: CRISPR-associated protein Csm2
    • RNA: target RNA
    • Protein or peptide: CRISPR-associated protein Cas10
    • Protein or peptide: CRISPR-associated protein Csm5
KeywordsType III-A CRISPR-Cas / Csm / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


exonuclease activity / defense response to virus / endonuclease activity / RNA binding / ATP binding
Similarity search - Function
: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / : / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 ...: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / : / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / Cas10/Cmr2, second palm domain / : / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / HD domain / HD domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
CRISPR system Cms endoribonuclease Csm3 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm2 / CRISPR system Cms protein Csm5
Similarity search - Component
Biological speciesLactococcus lactis subsp. lactis (lactic acid bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsRai J / Sridhara S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM099604 United States
CitationJournal: Commun Biol / Year: 2022
Title: Structural and biochemical characterization of in vivo assembled Lactococcus lactis CRISPR-Csm complex.
Authors: Sagar Sridhara / Jay Rai / Charlisa Whyms / Hemant Goswami / Huan He / Walter Woodside / Michael P Terns / Hong Li /
Abstract: The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies ...The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle and is in addition regulated by cellular metabolites such as divalent metals and ATP. Recognition of the foreign or cognate target RNA (CTR) triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self or non-cognate target RNA (NTR) that differs from CTR only in its 3'-protospacer flanking sequence (3'-PFS). Here we employ electron cryomicroscopy (cryoEM), functional assays, and comparative cross-linking to study in vivo assembled mesophilic Lactococcus lactis Csm (LlCsm) at the three functional states: apo, the CTR- and the NTR-bound. Unlike previously studied Csm complexes, we observed binding of 3'-PFS to Csm in absence of bound ATP and analyzed the structures of the four RNA cleavage sites. Interestingly, comparative crosslinking results indicate a tightening of the Csm3-Csm4 interface as a result of CTR but not NTR binding, reflecting a possible role of protein dynamics change during activation.
History
DepositionJul 2, 2020-
Header (metadata) releaseJan 12, 2022-
Map releaseJan 12, 2022-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0194
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0194
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xn4
  • Surface level: 0.0194
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22267.png

Downloads & links

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Map

FileDownload / File: emd_22267.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 274.944 Å		1.07 Å/pix.
x 256 pix.
= 274.944 Å		1.07 Å/pix.
x 256 pix.
= 274.944 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.074 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0194 / Movie #1: 0.0194
Minimum - Maximum-0.03230697 - 0.08447872
Average (Standard dev.)0.00045416332 (±0.003110726)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 274.944 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0741.0741.074
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z274.944274.944274.944
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0320.0840.000

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Supplemental data

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Sample components

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Entire : Lactococcus lactis Csm CRISPR-Cas CTR_3:2 Complex

EntireName: Lactococcus lactis Csm CRISPR-Cas CTR_3:2 Complex
Components
  • Complex: Lactococcus lactis Csm CRISPR-Cas CTR_3:2 Complex
    • Protein or peptide: CRISPR-associated protein Csm4
    • Protein or peptide: CRISPR-associated protein Csm3
    • RNA: Crispr RNA
    • Protein or peptide: CRISPR-associated protein Csm2
    • RNA: target RNA
    • Protein or peptide: CRISPR-associated protein Cas10
    • Protein or peptide: CRISPR-associated protein Csm5

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Supramolecule #1: Lactococcus lactis Csm CRISPR-Cas CTR_3:2 Complex

SupramoleculeName: Lactococcus lactis Csm CRISPR-Cas CTR_3:2 Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)

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Macromolecule #1: CRISPR-associated protein Csm4

MacromoleculeName: CRISPR-associated protein Csm4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 33.844148 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKIIKLYFES PVHFGEKRLS ESKITFSADT LFSALMIEAV GLGKEDEFYQ LASNNLVKFS DAFPFIDQYY YIPKPMFNLK LEKEDENPS KAFKKLLYVP IDSLEDYLSG GLDAYFERES FNLGKLALSE KVQQHDFKDS EPYNVGTFTF KENTGLYVLI E QTHPLLEE ...String:
MKIIKLYFES PVHFGEKRLS ESKITFSADT LFSALMIEAV GLGKEDEFYQ LASNNLVKFS DAFPFIDQYY YIPKPMFNLK LEKEDENPS KAFKKLLYVP IDSLEDYLSG GLDAYFERES FNLGKLALSE KVQQHDFKDS EPYNVGTFTF KENTGLYVLI E QTHPLLEE LLENLQYSGI GGKRNSGYGK FKFEILEDSD IEDLFSAKGN RKILLSGALP KDAELEQALK NASYLLERRG GF VQSDTYA TNLVKKQDLY VFKSGSTFEN SFDGDIYQVG KKGNHPVYKY AKSFFLEVS

UniProtKB: CRISPR system Cms protein Csm4

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Macromolecule #2: CRISPR-associated protein Csm3

MacromoleculeName: CRISPR-associated protein Csm3 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 23.82516 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKLVIEGTIV LKTGMHIGGS SDFSAIGAVA SPVVRDTLTR LPLIPGSSLK GKMRYLLAKE LNNGILLNEP NNDQDEILRL FGSSEKDKI RRARLKFNDI KLSNLAELET FNVSSTEVKF ENTINRKTAV ANPRQIERVI AGSKFDFEIF YNLDDIKEVE K DFENIKQG ...String:
MKLVIEGTIV LKTGMHIGGS SDFSAIGAVA SPVVRDTLTR LPLIPGSSLK GKMRYLLAKE LNNGILLNEP NNDQDEILRL FGSSEKDKI RRARLKFNDI KLSNLAELET FNVSSTEVKF ENTINRKTAV ANPRQIERVI AGSKFDFEIF YNLDDIKEVE K DFENIKQG FDLLEFDYLG GHGTRGSGRI AFENLSVITA VGNFEKINTL NEILGA

UniProtKB: CRISPR system Cms endoribonuclease Csm3

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Macromolecule #4: CRISPR-associated protein Csm2

MacromoleculeName: CRISPR-associated protein Csm2 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 15.796016 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
TELKIGNEKV NSTNFGDFAE KAIRGINHKP FVNSKGGEQK ITTSKIRGIL ELVNKVYNRV INTNDVELSE NILADIAYIK VKIAYESGR EPVVKDFIQR TAFTAAITDV MNQRTRESFL LFARYVESLI AYFKFYGGKD

UniProtKB: CRISPR system Cms protein Csm2

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Macromolecule #6: CRISPR-associated protein Cas10

MacromoleculeName: CRISPR-associated protein Cas10 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 87.132586 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDKINLVCGS LLHNIGKIIY RGTSERAKHS KLGGDFIKSF EQFRNTELTD CIRYHHAQEI TSVKSNKEKN SLFYITYIAD NISSGMDRR KDLEEGAEGF NWDKKVALGS VFNVLNEKEK GRQNYSYPFV ARTRIKEEPL NFPTATQNQY TTSYYDGLIT D MKTILQRL ...String:
MDKINLVCGS LLHNIGKIIY RGTSERAKHS KLGGDFIKSF EQFRNTELTD CIRYHHAQEI TSVKSNKEKN SLFYITYIAD NISSGMDRR KDLEEGAEGF NWDKKVALGS VFNVLNEKEK GRQNYSYPFV ARTRIKEEPL NFPTATQNQY TTSYYDGLIT D MKTILQRL KPDKEHINSL LQMMESLWSY VPSSTDKNQL VDISLYDHSR TTAAIASAIY DYFQAENITD YQKELFDYNA TE FYDKNAF LMMNFDMSGV QNFIYNISGS KALKSLRARS FYLDMLLEYI SDNLLEKLEL SRANILYVGG GHAYLLLANT NKT KAILSD FEHDLKTWFL DKFKIDLYVA MAYTEVSAND LMNHNGHYRD IYRRLSQKTS AKKANRYTAE EILNLNHQGT ENAR ECREC KRSDLLIEED DICEICDSLQ KVSRDLTREN IFVIANEGVL DMPFGKKMSA LSYSQADKLK KSNAEVQIYA KNISE IGQN LMTRIDMGDY TYRSDFHEML EEVEVGINRL GVLRADVDNL GQAFINGIPD DYLSISRTAT FSRAMSRFFK NYLNQL LAE KSYKINVIYA GGDDLFMIGA WQDILDFSIV LKQKFADFTQ NKLSISAGIG MFREKYPVAR MASLTGDLED AAKDYKP DE RAVQATKNAV TLFDATNVFS WDTLENDIFV KLDAITKNFE KLDETGKAFI YRLIDLLRGV NENQQINIAR LAYTLSRM E EKIGKTFAQE LYNWANADRK TLIMALEIYI LKTRE

UniProtKB: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)

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Macromolecule #7: CRISPR-associated protein Csm5

MacromoleculeName: CRISPR-associated protein Csm5 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 40.492617 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKTYRVTLT ALGPIFIGGG EKLKKYEYIF DKQKKVAHMI DHTKFTKYLL EKNLLDDFTS RVNSHFDLYD YLVNKKGIVF MPLVKYSVP VAQFRTEVKN RFGKPISSPP MNDLNTFVKD AFGRPYIPGS SLKGALRTAI LNDLKEDTKE NEVFAHLQVS D SETIDLEN ...String:
MKKTYRVTLT ALGPIFIGGG EKLKKYEYIF DKQKKVAHMI DHTKFTKYLL EKNLLDDFTS RVNSHFDLYD YLVNKKGIVF MPLVKYSVP VAQFRTEVKN RFGKPISSPP MNDLNTFVKD AFGRPYIPGS SLKGALRTAI LNDLKEDTKE NEVFAHLQVS D SETIDLEN LKVYQKVDYS KTAKPLPLYR ECLKPNTEIT FTVSFDDEYL TLKKIQNALH KTYQHYYIKW LKGGKVGETL IK GVYDSHA DELKKNTFAL DQPSQNQGEI IYIGGGAGFV SKTLHYKSKN RDQARNDSFD ILKQLFRTTY SKMRSVPDNV PVA LKLAVE TKTFNGRVTG KHYLEMGKAR IKLEEL

UniProtKB: CRISPR system Cms protein Csm5

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Macromolecule #3: Crispr RNA

MacromoleculeName: Crispr RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 11.133669 KDa
SequenceString:
ACGAGAACAU ACGUUCUUUG AACCAAGCUU CAACU

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Macromolecule #5: target RNA

MacromoleculeName: target RNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 9.678801 KDa
SequenceString:
GUUGAAGCUU GGUUCAAAGA ACGUAUCAAG

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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
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.14 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: OTHER / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 62413
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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