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- PDB-7v01: Staphylococcus epidermidis RP62a CRISPR short effector complex wi... -

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Basic information

Entry
Database: PDB / ID: 7v01
TitleStaphylococcus epidermidis RP62a CRISPR short effector complex with self RNA target and ATP
Components
  • (CRISPR system ...) x 5
  • (RNA (37-MER)) x 2
KeywordsHYDROLASE/RNA / Type IIIA CRISPR / effector complex / RNA binding protein / HYDROLASE-RNA complex
Function / homology
Function and homology information


exonuclease activity / endonuclease activity / defense response to virus / hydrolase activity / RNA binding / ATP binding / metal ion 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 / HD domain / GGDEF domain profile. / GGDEF domain / HD domain / Reverse transcriptase/Diguanylate cyclase domain / Reverse transcriptase/Diguanylate cyclase domain / Alpha-Beta Plaits / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / RNA / RNA (> 10) / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm2 / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm5
Similarity search - Component
Biological speciesStaphylococcus epidermidis RP62A (bacteria)
Staphylococcus epidermidis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.67 Å
AuthorsSmith, E.M. / Ferrell, S.H. / Tokars, V.L. / Mondragon, A.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM118108 United States
American Cancer Society134255-PF-20-041-01-DMC United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM129541 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM129539 United States
CitationJournal: Structure / Year: 2022
Title: Structures of an active type III-A CRISPR effector complex.
Authors: Eric M Smith / Sé Ferrell / Valerie L Tokars / Alfonso Mondragón /
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) provide many prokaryotes with an adaptive immune system against invading genetic material. ...Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) provide many prokaryotes with an adaptive immune system against invading genetic material. Type III CRISPR systems are unique in that they can degrade both RNA and DNA. In response to invading nucleic acids, they produce cyclic oligoadenylates that act as secondary messengers, activating cellular nucleases that aid in the immune response. Here, we present seven single-particle cryo-EM structures of the type III-A Staphylococcus epidermidis CRISPR effector complex. The structures reveal the intact S. epidermidis effector complex in an apo, ATP-bound, cognate target RNA-bound, and non-cognate target RNA-bound states and illustrate how the effector complex binds and presents crRNA. The complexes bound to target RNA capture the type III-A effector complex in a post-RNA cleavage state. The ATP-bound structures give details about how ATP binds to Cas10 to facilitate cyclic oligoadenylate production.
History
DepositionMay 9, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 6, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 17, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 16, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR system Cms endoribonuclease Csm3
B: CRISPR system Cms endoribonuclease Csm3
C: CRISPR system Cms endoribonuclease Csm3
E: CRISPR system Cms protein Csm5
F: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
G: RNA (37-MER)
H: CRISPR system Cms protein Csm4
I: CRISPR system Cms protein Csm2
K: CRISPR system Cms protein Csm2
U: RNA (37-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)289,30612
Polymers288,29110
Non-polymers1,0142
Water00
1


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

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Components

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CRISPR system ... , 5 types, 8 molecules ABCEFHIK

#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 natural source
Source: (natural) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A / References: UniProt: Q5HK91
#2: 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 natural source
Source: (natural) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A / References: UniProt: Q5HK93
#3: Protein CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / Cyclic oligoadenylate synthase


Mass: 87685.781 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A / References: UniProt: Q5HK89
#5: Protein CRISPR system Cms protein Csm4


Mass: 34551.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A / References: UniProt: Q5HK92
#6: Protein CRISPR system Cms protein Csm2 / CRISPR type III A-associated protein Csm2


Mass: 15436.794 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A / References: UniProt: Q5HK90

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RNA chain , 2 types, 2 molecules GU

#4: RNA chain RNA (37-MER)


Mass: 11895.168 Da / Num. of mol.: 1
Fragment: Staphylococcus epidermidis RP62A CRISPR RNA: Repeat plus Spacer sequence 1
Source method: isolated from a natural source
Source: (natural) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A
#7: RNA chain RNA (37-MER)


Mass: 11733.888 Da / Num. of mol.: 1 / Fragment: CRISPR self RNA target / Source method: obtained synthetically / Source: (synth.) Staphylococcus epidermidis (bacteria)

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Non-polymers , 1 types, 2 molecules

#8: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM

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Details

Has ligand of interestY
Has protein modificationY

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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: Staphylococcus epidermidis RP62a CRISPR short effector complex with self target RNA and ATP
Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Staphylococcus epidermidis (bacteria) / Strain: RP62A
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM2-Amino-2-(hydroxymethyl)propane-1,3-diolC4H11NO31
2250 mMNaClNaCl1
31 mMDithiothreitolC4H10O2S21
SpecimenConc.: 3.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The purified complex was mixed with 1 mM ATP. This complex was allowed to incubate for 10 minutes ice before being dialyzed (Slide-A-Lyzer MINI Dialysis Devices, Thermo Fisher) overnight in ...Details: The purified complex was mixed with 1 mM ATP. This complex was allowed to incubate for 10 minutes ice before being dialyzed (Slide-A-Lyzer MINI Dialysis Devices, Thermo Fisher) overnight in 50 mM Tris pH 8.0, 250 mM NaCl, 1 mM DTT at 4 C. Prior to vitrification the complex was mixed in a 1:10 molar ratio with a 37 nt RNA that was completely complementary to the 37 nt version of crRNA 1. Additional ATP was added to the incubated ribonucleoprotein complex to a final concentration of 2 mM and allowed to incubate on ice for 15 minutes.
Specimen supportDetails: cryo-EM grids were prepared by glow discharging for 10 seconds at 15 mA in a Pelco easiGlow glow discharger.
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 46772 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 52.79 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 7527

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
PDB_EXTRACT3.27data extraction
EM software
IDNameVersionCategory
2Leginonimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9PHENIX1.18.2model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1790337
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 130430 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
16NBT

6nbt

A6NBT1
26NBT

6nbt

B6NBT1
36NBT

6nbt

C6NBT1
46NBU

6nbu

I6NBU2
56NBU

6nbu

K6NBU2
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 216.49 Å2 / Biso mean: 99.426 Å2 / Biso min: 1.16 Å2

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