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- EMDB-43814: Cryo-EM structure of the active Lactococcus lactis Csm bound to t... -

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Basic information

Entry
Database: EMDB / ID: EMD-43814
TitleCryo-EM structure of the active Lactococcus lactis Csm bound to target in post-cleavage stage
Map data
Sample
  • Complex: ATP bound Lactococcus lactis Csm CRISPR-Cas complex with stable Cas10
    • Protein or peptide: x 5 types
    • RNA: x 3 types
  • Ligand: x 3 types
KeywordsType III-A CRISPR-Cas / Csm / Cyclic Oligoadenylate synthesis / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


exonuclease activity / endonuclease activity / defense response to virus / 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 / : / 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: 2.58 Å
AuthorsWang B / Goswami HN / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM099604 United States
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Molecular basis for cA6 synthesis by a type III-A CRISPR-Cas enzyme and its conversion to cA4 production.
Authors: Hemant N Goswami / Fozieh Ahmadizadeh / Bing Wang / Doreen Addo-Yobo / Yu Zhao / A Carl Whittington / Huan He / Michael P Terns / Hong Li /
Abstract: The type III-A (Csm) CRISPR-Cas systems are multi-subunit and multipronged prokaryotic enzymes in guarding the hosts against viral invaders. Beyond cleaving activator RNA transcripts, Csm confers ...The type III-A (Csm) CRISPR-Cas systems are multi-subunit and multipronged prokaryotic enzymes in guarding the hosts against viral invaders. Beyond cleaving activator RNA transcripts, Csm confers two additional activities: shredding single-stranded DNA and synthesizing cyclic oligoadenylates (cOAs) by the Cas10 subunit. Known Cas10 enzymes exhibit a fascinating diversity in cOA production. Three major forms-cA3, cA4 and cA6have been identified, each with the potential to trigger unique downstream effects. Whereas the mechanism for cOA-dependent activation is well characterized, the molecular basis for synthesizing different cOA isoforms remains unclear. Here, we present structural characterization of a cA6-producing Csm complex during its activation by an activator RNA. Analysis of the captured intermediates of cA6 synthesis suggests a 3'-to-5' nucleotidyl transferring process. Three primary adenine binding sites can be identified along the chain elongation path, including a unique tyrosine-threonine dyad found only in the cA6-producing Cas10. Consistently, disrupting the tyrosine-threonine dyad specifically impaired cA6 production while promoting cA4 production. These findings suggest that Cas10 utilizes a unique enzymatic mechanism for forming the phosphodiester bond and has evolved distinct strategies to regulate the cOA chain length.
History
DepositionFeb 25, 2024-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateAug 14, 2024-
Current statusAug 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_43814.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.074 Å
Density
Contour LevelBy AUTHOR: 0.677
Minimum - Maximum-4.0078297 - 7.8922076
Average (Standard dev.)-0.000028275497 (±0.13440803)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 378.048 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : ATP bound Lactococcus lactis Csm CRISPR-Cas complex with stable Cas10

EntireName: ATP bound Lactococcus lactis Csm CRISPR-Cas complex with stable Cas10
Components
  • Complex: ATP bound Lactococcus lactis Csm CRISPR-Cas complex with stable Cas10
    • Protein or peptide: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
    • Protein or peptide: CRISPR-associated protein Csm4
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • RNA: CRISPR RNA
    • Protein or peptide: CRISPR system Cms protein Csm2
    • RNA: Target RNA
    • Protein or peptide: CRISPR system Cms protein Csm5
    • RNA: RNA (5'-D(*(ATP))-R(P*AP*A)-3')
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: ATP bound Lactococcus lactis Csm CRISPR-Cas complex with stable Cas10

SupramoleculeName: ATP bound Lactococcus lactis Csm CRISPR-Cas complex with stable Cas10
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)

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Macromolecule #1: 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: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 87.365852 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDKINLVCGS LLADIGKIIY RGTSERAKHS KLGGDFIKSF EQFRNTELTD CIRYHHAQEI TSVKSNKEKN SLFYITYIAD NISSGMDRR KDLEEGAEGF NWDKKVALGS VFNVLNEKEK GRQNYSYPFV ARTRIKEEPL NFPTATQNQY TTSYYDGLIT D MKTILQRL ...String:
MDKINLVCGS LLADIGKIIY 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 LKTRERAA

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

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Macromolecule #2: CRISPR-associated protein Csm4

MacromoleculeName: CRISPR-associated protein Csm4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 33.943277 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 AKSFFLEVSV

UniProtKB: CRISPR system Cms protein Csm4

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Macromolecule #3: CRISPR system Cms endoribonuclease Csm3

MacromoleculeName: CRISPR system Cms endoribonuclease Csm3 / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 23.86917 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKLVIEGTIV LKTGMHIGGS SDFSAIGAVD SPVVRDTLTR LPLIPGSSLK GKMRYLLAKE LNNGILLNEP NNDQDEILRL FGSSEKDKI RRARLKFNDI KLSNLAELET FNVSSTEVKF ENTINRKTAV ANPRQIERVI AGSKFDFEIF YNLDDIKEVE K DFENIKQG ...String:
MKLVIEGTIV LKTGMHIGGS SDFSAIGAVD 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 #5: CRISPR system Cms protein Csm2

MacromoleculeName: CRISPR system Cms protein Csm2 / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 17.01433 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGHHHHHHSG GTELKIGNEK VNSTNFGDFA EKAIRGINHK PFVNSKGGEQ KITTSKIRGI LELVNKVYNR VINTNDVELS ENILADIAY IKVKIAYESG REPVVKDFIQ RTAFTAAITD VMNQRTRESF LLFARYVESL IAYFKFYGGK D

UniProtKB: CRISPR system Cms protein Csm2

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Macromolecule #7: CRISPR system Cms protein Csm5

MacromoleculeName: CRISPR system Cms 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: 40.621797 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 IKLEELK

UniProtKB: CRISPR system Cms protein Csm5

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Macromolecule #4: CRISPR RNA

MacromoleculeName: CRISPR RNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 11.946271 KDa
SequenceString:
ACGAGAACGC AGCACCAGCU GUCCAACCUG AAGAAGA

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Macromolecule #6: Target RNA

MacromoleculeName: Target RNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 11.558874 KDa
SequenceString:
CUUCUUCAGG UUGGACAGCU GGUGCUGCCA AGAGCA

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Macromolecule #8: RNA (5'-D(*(ATP))-R(P*AP*A)-3')

MacromoleculeName: RNA (5'-D(*(ATP))-R(P*AP*A)-3') / type: rna / ID: 8 / Number of copies: 1
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Molecular weightTheoretical: 1.10262 KDa
SequenceString:
(ATP)AA

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Macromolecule #9: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 9 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #11: water

MacromoleculeName: water / type: ligand / ID: 11 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 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: 2.58 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 965374
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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