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- EMDB-27762: 318 kDa Cas10-Csm effector complex bound to cognate target RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-27762
Title318 kDa Cas10-Csm effector complex bound to cognate target RNA
Map data318 kDa Cas10-Csm effector complex
Sample
  • Complex: 318 kDa Cas10-Csm effector complex bound to cognate target RNA
    • Protein or peptide: Csm2
    • Protein or peptide: Csm3
    • Protein or peptide: Csm4
    • Protein or peptide: Csm5
    • RNA: crRNA
    • RNA: cognate target RNA
    • Protein or peptide: Cas10
KeywordsCRISPR / Cas10 / Type III / Csm / RNA BINDING PROTEIN
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
DepositionAug 2, 2022-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27762.png

Downloads & links

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Map

FileDownload / File: emd_27762.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation318 kDa Cas10-Csm effector complex
Voxel sizeX=Y=Z: 0.846 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-1.0226691 - 1.7918974
Average (Standard dev.)-0.00023839192 (±0.03885111)
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: #2

Fileemd_27762_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_27762_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 : 318 kDa Cas10-Csm effector complex bound to cognate target RNA

EntireName: 318 kDa Cas10-Csm effector complex bound to cognate target RNA
Components
  • Complex: 318 kDa Cas10-Csm effector complex bound to cognate target RNA
    • Protein or peptide: Csm2
    • Protein or peptide: Csm3
    • Protein or peptide: Csm4
    • Protein or peptide: Csm5
    • RNA: crRNA
    • RNA: cognate target RNA
    • Protein or peptide: Cas10

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Supramolecule #1: 318 kDa Cas10-Csm effector complex bound to cognate target RNA

SupramoleculeName: 318 kDa Cas10-Csm effector complex bound to cognate target RNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
Molecular weightTheoretical: 318 KDa

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Macromolecule #1: Csm2

MacromoleculeName: Csm2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString:
MTFAHEVVKS NVKNVKDRKG KEKQVLFNGL TTSKLRNLME QVNRLYTIAF NSNEDQLNEE FIDELEYLK IKFYYEAGRE KSVDEFLKKT LMFPIIDRVI KKESKKFFLD YCKYFEALVA Y AKYYQKED

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Macromolecule #2: Csm3

MacromoleculeName: Csm3 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
SequenceString: MYSKIKISGT IEVVTGLHIG GGGESSMIGA IDSPVVRDLQ TKLPIIPGSS IKGKMRNLLA KHFGLKMKQE SHNQDDERVL RLFGSSEKGN IQRARLQISD AFFSEKTKEH FAQNDIAYTE TKFENTINRL TAVANPRQIE RVTRGSEFDF VFIYNVDEES QVEDDFENIE ...String:
MYSKIKISGT IEVVTGLHIG GGGESSMIGA IDSPVVRDLQ TKLPIIPGSS IKGKMRNLLA KHFGLKMKQE SHNQDDERVL RLFGSSEKGN IQRARLQISD AFFSEKTKEH FAQNDIAYTE TKFENTINRL TAVANPRQIE RVTRGSEFDF VFIYNVDEES QVEDDFENIE KAIHLLENDY LGGGGTRGNG RIQFKDTNIE TVVGEYDSTN LKI

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Macromolecule #3: Csm4

MacromoleculeName: Csm4 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
SequenceString: MTLATKVFKL SFKTPVHFGK KRLSDGEMTI TADTLFSALF IETLQLGKDT DWLLNDLIIS DTFPYENELY YLPKPLIKID SKEEDNHKAF KKLKYVPVHH YNQYLNGELS AEDATDLNDI FNIGYFSLQT KVSLIAQETD SSADSEPYSV GTFTFEPEAG LYFIAKGSEE ...String:
MTLATKVFKL SFKTPVHFGK KRLSDGEMTI TADTLFSALF IETLQLGKDT DWLLNDLIIS DTFPYENELY YLPKPLIKID SKEEDNHKAF KKLKYVPVHH YNQYLNGELS AEDATDLNDI FNIGYFSLQT KVSLIAQETD SSADSEPYSV GTFTFEPEAG LYFIAKGSEE TLDHLNNIMT ALQYSGLGGK RNAGYGQFEY EIINNQQLSK LLNQNGKHSI LLSTAMAKKE EIESALKEAR YILTKRSGFV QSTNYSEMLV KKSDFYSFSS GSVFKNIFNG DIFNVGHNGK HPVYRYAKPL WLEV

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Macromolecule #4: Csm5

MacromoleculeName: Csm5 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
SequenceString: MTIKNYEVVI KTLGPIHIGS GQVMKKQDYI YDFYNSKVYM INGNKLVKFL KRKNLLYTYQ NFLRYPPKNP RENGLKDYLD AQNVKQSEWE AFVSYSEKVN QGKKYGNTRP KPLNDLHLMV RDGQNKVYLP GSSIKGAIKT TLVSKYNNEK NKDIYSKIKV SDSKPIDESN ...String:
MTIKNYEVVI KTLGPIHIGS GQVMKKQDYI YDFYNSKVYM INGNKLVKFL KRKNLLYTYQ NFLRYPPKNP RENGLKDYLD AQNVKQSEWE AFVSYSEKVN QGKKYGNTRP KPLNDLHLMV RDGQNKVYLP GSSIKGAIKT TLVSKYNNEK NKDIYSKIKV SDSKPIDESN LAIYQKIDIN KSEKSMPLYR ECIDVNTEIK FKLTIEDEIY SINEIEQSIQ DFYKNYYDKW LVGFKETKGG RRFALEGGIP DVLNQNILFL GAGTGFVSKT THYQLKNRKQ AKQDSFEILT KKFRGTYGKM KEIPSNVPVA LKGTTNQSRH TSYQQGMCKV SFQELNNEVL

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Macromolecule #7: Cas10

MacromoleculeName: Cas10 / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
SequenceString: MNKKNILMYG SLLHDIGKII YRSGDHTFSR GTHSKLGHQF LSQFSEFKDN EVLDNVAYHH YKELAKANL DNDNTAYITY IADNIASGID RRDIIEEGDE EYEKQLFNFD KYTPLYSVFN I VNSEKLKQ TNGKFKFSNE SNIEYPKTEN IQYSSGNYTT LMKDMSHDLE ...String:
MNKKNILMYG SLLHDIGKII YRSGDHTFSR GTHSKLGHQF LSQFSEFKDN EVLDNVAYHH YKELAKANL DNDNTAYITY IADNIASGID RRDIIEEGDE EYEKQLFNFD KYTPLYSVFN I VNSEKLKQ TNGKFKFSNE SNIEYPKTEN IQYSSGNYTT LMKDMSHDLE HKLSIKEGTF PS LLQWTES LWQYVPSSTN KNQLIDISLY DHSRITCAIA SCIFDYLNEN NIHNYKDELF SKY ENTKSF YQKEAFLLLS MDMSGIQDFI YNISGSKALK SLRSRSFYLE LMLEVIVDQL LERL ELARA NLLYTGGGHA YLLVSNTDKV KKKITQFNNE LKKWFMSEFT TDLSLSMAFE KCSGD DLMN TSGNYRTIWR NVSSKLSDIK AHKYSAEDIL KLNHFHSYGD RECKECLRSD IDINDD GLC SICEGIINIS NDLRDKSFFV LSETGKLKMP FNKFISVIDY EEAEMLVQNN NQVRIYS KN KPYIGIGIST NLWMCDYDYA SQNQDMREKG IGSYVDREEG VKRLGVVRAD IDNLGATF I SGIPEKYNSI SRTATLSRQL SLFFKYELNH LLENYQITAI YSGGDDLFLI GAWDDIIEA SIYINDKFKE FTLDKLTLSA GVGMFSGKYP VSKMAFETGR LEEAAKTGEK NQISLWLQEK VYNWDEFKK NILEEKLLVL QQGFSQTDEH GKAFIYKMLA LLRNNEAINI ARLAYLLARS K MNEDFTSK IFNWAQNDKD KNQLITALEY YIYQIREAD

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Macromolecule #5: crRNA

MacromoleculeName: crRNA / type: rna / ID: 5
SequenceString:
ACGAGAACAC GUAUGCCGAA GUAUAUAAAU CAUCAGUACA AAG

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Macromolecule #6: cognate target RNA

MacromoleculeName: cognate target RNA / type: rna / ID: 6
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: 164000
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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