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- EMDB-27593: The structure of S. epidermidis Cas10-Csm bound to target RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-27593
TitleThe structure of S. epidermidis Cas10-Csm bound to target RNA
Map dataCas10-Csm complex bound to crRNA and target RNA.
Sample
  • Complex: Cas10-Csm complex bound to target RNA
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • RNA: crRNA
    • RNA: Target RNA
    • Protein or peptide: CRISPR system Cms protein Csm2
    • Protein or peptide: CRISPR system Cms protein Csm4
    • Protein or peptide: CRISPR system Cms protein Csm5
KeywordsCRISPR / Cas10 / Type III / Csm / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


endonuclease activity / defense response to virus / RNA binding / metal ion binding
Similarity search - Function
Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein Csm5 / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR type III-associated protein / RAMP superfamily
Similarity search - Domain/homology
: / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm5
Similarity search - Component
Biological speciesStaphylococcus epidermidis RP62A (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsParaan M / Stagg SM / Dunkle JA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM142966 United States
CitationJournal: PLoS One / Year: 2023
Title: The structure of a Type III-A CRISPR-Cas effector complex reveals conserved and idiosyncratic contacts to target RNA and crRNA among Type III-A systems.
Authors: Mohammadreza Paraan / Mohamed Nasef / Lucy Chou-Zheng / Sarah A Khweis / Allyn J Schoeffler / Asma Hatoum-Aslan / Scott M Stagg / Jack A Dunkle /
Abstract: Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the ...Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 Å density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.
History
DepositionJul 12, 2022-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27593.png

Downloads & links

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Map

FileDownload / File: emd_27593.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas10-Csm complex bound to crRNA and target RNA.
Voxel sizeX=Y=Z: 0.846 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.3015177 - 1.885284
Average (Standard dev.)-0.0002685595 (±0.039473563)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 338.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_27593_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_27593_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas10-Csm complex bound to target RNA

EntireName: Cas10-Csm complex bound to target RNA
Components
  • Complex: Cas10-Csm complex bound to target RNA
    • Protein or peptide: CRISPR system Cms endoribonuclease Csm3
    • RNA: crRNA
    • RNA: Target RNA
    • Protein or peptide: CRISPR system Cms protein Csm2
    • Protein or peptide: CRISPR system Cms protein Csm4
    • Protein or peptide: CRISPR system Cms protein Csm5

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Supramolecule #1: Cas10-Csm complex bound to target RNA

SupramoleculeName: Cas10-Csm complex bound to target RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
Molecular weightTheoretical: 300 KDa

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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: 3 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria) / Strain: ATCC 35984 / RP62A
Molecular weightTheoretical: 24.033975 KDa
Recombinant expressionOrganism: Staphylococcus epidermidis RP62A (bacteria)
SequenceString: MYSKIKISGT IEVVTGLHIG GGGESSMIGA IDSPVVRDLQ TKLPIIPGSS IKGKMRNLLA KHFGLKMKQE SHNQDDERVL RLFGSSEKG NIQRARLQIS DAFFSEKTKE HFAQNDIAYT ETKFENTINR LTAVANPRQI ERVTRGSEFD FVFIYNVDEE S QVEDDFEN ...String:
MYSKIKISGT IEVVTGLHIG GGGESSMIGA IDSPVVRDLQ TKLPIIPGSS IKGKMRNLLA KHFGLKMKQE SHNQDDERVL RLFGSSEKG NIQRARLQIS DAFFSEKTKE HFAQNDIAYT ETKFENTINR LTAVANPRQI ERVTRGSEFD FVFIYNVDEE S QVEDDFEN IEKAIHLLEN DYLGGGGTRG NGRIQFKDTN IETVVGEYDS TNLKIK

UniProtKB: CRISPR system Cms endoribonuclease Csm3

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Macromolecule #4: CRISPR system Cms protein Csm2

MacromoleculeName: CRISPR system Cms protein Csm2 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
Molecular weightTheoretical: 16.809471 KDa
Recombinant expressionOrganism: Staphylococcus epidermidis RP62A (bacteria)
SequenceString:
MILAKTKSGK TIDLTFAHEV VKSNVKNVKD RKGKEKQVLF NGLTTSKLRN LMEQVNRLYT IAFNSNEDQL NEEFIDELEY LKIKFYYEA GREKSVDEFL KKTLMFPIID RVIKKESKKF FLDYCKYFEA LVAYAKYYQK ED

UniProtKB: UNIPROTKB: A0A8G7QML1

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Macromolecule #5: CRISPR system Cms protein Csm4

MacromoleculeName: CRISPR system Cms protein Csm4 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria) / Strain: ATCC 35984 / RP62A
Molecular weightTheoretical: 34.551938 KDa
Recombinant expressionOrganism: Staphylococcus epidermidis RP62A (bacteria)
SequenceString: MTLATKVFKL SFKTPVHFGK KRLSDGEMTI TADTLFSALF IETLQLGKDT DWLLNDLIIS DTFPYENELY YLPKPLIKID SKEEDNHKA FKKLKYVPVH HYNQYLNGEL SAEDATDLND IFNIGYFSLQ TKVSLIAQET DSSADSEPYS VGTFTFEPEA G LYFIAKGS ...String:
MTLATKVFKL SFKTPVHFGK KRLSDGEMTI TADTLFSALF IETLQLGKDT DWLLNDLIIS DTFPYENELY YLPKPLIKID SKEEDNHKA FKKLKYVPVH HYNQYLNGEL SAEDATDLND IFNIGYFSLQ TKVSLIAQET DSSADSEPYS VGTFTFEPEA G LYFIAKGS EETLDHLNNI MTALQYSGLG GKRNAGYGQF EYEIINNQQL SKLLNQNGKH SILLSTAMAK KEEIESALKE AR YILTKRS GFVQSTNYSE MLVKKSDFYS FSSGSVFKNI FNGDIFNVGH NGKHPVYRYA KPLWLEV

UniProtKB: CRISPR system Cms protein Csm4

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Macromolecule #6: CRISPR system Cms protein Csm5

MacromoleculeName: CRISPR system Cms protein Csm5 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria) / Strain: ATCC 35984 / RP62A
Molecular weightTheoretical: 39.449125 KDa
Recombinant expressionOrganism: Staphylococcus epidermidis RP62A (bacteria)
SequenceString: MTIKNYEVVI KTLGPIHIGS GQVMKKQDYI YDFYNSKVYM INGNKLVKFL KRKNLLYTYQ NFLRYPPKNP RENGLKDYLD AQNVKQSEW EAFVSYSEKV NQGKKYGNTR PKPLNDLHLM VRDGQNKVYL PGSSIKGAIK TTLVSKYNNE KNKDIYSKIK V SDSKPIDE ...String:
MTIKNYEVVI KTLGPIHIGS GQVMKKQDYI YDFYNSKVYM INGNKLVKFL KRKNLLYTYQ NFLRYPPKNP RENGLKDYLD AQNVKQSEW EAFVSYSEKV NQGKKYGNTR PKPLNDLHLM VRDGQNKVYL PGSSIKGAIK TTLVSKYNNE KNKDIYSKIK V SDSKPIDE SNLAIYQKID INKSEKSMPL YRECIDVNTE IKFKLTIEDE IYSINEIEQS IQDFYKNYYD KWLVGFKETK GG RRFALEG GIPDVLNQNI LFLGAGTGFV SKTTHYQLKN RKQAKQDSFE ILTKKFRGTY GKMKEIPSNV PVALKGTTNQ SRH TSYQQG MCKVSFQELN NEVL

UniProtKB: CRISPR system Cms protein Csm5

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
Molecular weightTheoretical: 11.895168 KDa
SequenceString:
ACGAGAACAC GUAUGCCGAA GUAUAUAAAU CAUCAGU

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Macromolecule #3: Target RNA

MacromoleculeName: Target RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
Molecular weightTheoretical: 13.599979 KDa
SequenceString:
CUUUGUACUG AUGAUUUAUA UACUUCGGCA UACGUUCUCU AAA

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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 / Component:
ConcentrationName
50.0 mMTris-HCl
150.0 mMNaCl
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 78.0 K / Max: 92.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 5000 / Average exposure time: 1.0 sec. / Average electron dose: 44.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 1.5 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 81000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 0.001 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsAll processing was done in cryoSPARC 2.
Particle selectionNumber selected: 1400000 / Details: Using Topaz as implemented in cryoSPARC.
Startup modelType of model: OTHER / Details: ab initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 122000
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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