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- PDB-8do6: The structure of S. epidermidis Cas10-Csm bound to target RNA -

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Basic information

Entry
Database: PDB / ID: 8do6
TitleThe structure of S. epidermidis Cas10-Csm bound to target RNA
Components
  • (CRISPR system Cms protein ...) x 3
  • CRISPR system Cms endoribonuclease Csm3
  • Target RNA
  • crRNA
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / Cas10 / Type III / Csm / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


defense response to virus / endonuclease activity / RNA 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
RNA / RNA (> 10) / : / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm5
Similarity search - Component
Biological speciesStaphylococcus epidermidis RP62A (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsParaan, M. / Stagg, S.M. / Dunkle, J.A.
Funding support United States, 1items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: CRISPR system Cms endoribonuclease Csm3
G: CRISPR system Cms endoribonuclease Csm3
F: CRISPR system Cms endoribonuclease Csm3
I: crRNA
J: Target RNA
C: CRISPR system Cms protein Csm2
D: CRISPR system Cms protein Csm2
B: CRISPR system Cms protein Csm4
H: CRISPR system Cms protein Csm5


Theoretical massNumber of molelcules
Total (without water)205,2179
Polymers205,2179
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 3 molecules EGF

#1: Protein CRISPR system Cms endoribonuclease Csm3 / CRISPR type III A-associated RAMP protein Csm3


Mass: 24033.975 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A / Gene: SERP2459
Production host: Staphylococcus epidermidis RP62A (bacteria)
References: UniProt: Q5HK91

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RNA chain , 2 types, 2 molecules IJ

#2: RNA chain crRNA


Mass: 11895.168 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus epidermidis RP62A (bacteria)
Production host: Staphylococcus epidermidis RP62A (bacteria)
#3: RNA chain Target RNA


Mass: 13599.979 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Staphylococcus epidermidis RP62A (bacteria)

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CRISPR system Cms protein ... , 3 types, 4 molecules CDBH

#4: Protein CRISPR system Cms protein Csm2 / CRISPR type III A-associated protein Csm2


Mass: 16809.471 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus epidermidis RP62A (bacteria)
Gene: csm2, E1M97_06325, E1M99_06625
Production host: Staphylococcus epidermidis RP62A (bacteria)
References: UniProt: A0A8G7QML1
#5: Protein CRISPR system Cms protein Csm4


Mass: 34551.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A / Gene: SERP2458
Production host: Staphylococcus epidermidis RP62A (bacteria)
References: UniProt: Q5HK92
#6: Protein CRISPR system Cms protein Csm5 / CRISPR type III A-associated protein Csm5


Mass: 39449.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A / Gene: SERP2457
Production host: Staphylococcus epidermidis RP62A (bacteria)
References: UniProt: Q5HK93

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cas10-Csm complex bound to target RNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Staphylococcus epidermidis RP62A (bacteria)
Source (recombinant)Organism: Staphylococcus epidermidis (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMTris-HClTris1
2150 mMNaClSodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Calibrated magnification: 81000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 1500 nm / Cs: 0.001 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 92 K / Temperature (min): 78 K
Image recordingAverage exposure time: 1 sec. / Electron dose: 44.7 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5000
Image scansWidth: 5760 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameCategory
2Leginonimage acquisition
13cryoSPARC3D reconstruction
Image processingDetails: All processing was done in cryoSPARC 2.
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 1400000 / Details: Using Topaz as implemented in cryoSPARC.
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122000 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 71.07 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00513340
ELECTRON MICROSCOPYf_angle_d0.669618234
ELECTRON MICROSCOPYf_chiral_restr0.04272052
ELECTRON MICROSCOPYf_plane_restr0.00452120
ELECTRON MICROSCOPYf_dihedral_angle_d11.30712290

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