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- PDB-7uzy: Staphylococcus epidermidis RP62A CRISPR effector complex with non... -

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Basic information

Entry
Database: PDB / ID: 7uzy
TitleStaphylococcus epidermidis RP62A CRISPR effector complex with non-self target RNA 2
Components
  • (CRISPR system ...) x 4
  • RNA (37-MER)
  • RNA (40-MER)
KeywordsHYDROLASE/RNA / Type IIIA CRISPR / effector complex / RNA binding protein / HYDROLASE-RNA complex
Function / homology
Function and homology information


exonuclease activity / defense response to virus / endonuclease activity / 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, 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 ...: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / 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
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
Similarity search - Component
Biological speciesStaphylococcus epidermidis RP62A (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.05 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
F: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
J: CRISPR system Cms protein Csm2
I: CRISPR system Cms protein Csm2
K: CRISPR system Cms protein Csm2
D: CRISPR system Cms endoribonuclease Csm3
C: CRISPR system Cms endoribonuclease Csm3
A: CRISPR system Cms endoribonuclease Csm3
B: CRISPR system Cms endoribonuclease Csm3
H: CRISPR system Cms protein Csm4
G: RNA (37-MER)
L: RNA (40-MER)


Theoretical massNumber of molelcules
Total (without water)289,28511
Polymers289,28511
Non-polymers00
Water0
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 ... , 4 types, 9 molecules FJIKDCABH

#1: 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
#2: Protein CRISPR system Cms protein Csm2 / CRISPR type III A-associated protein Csm2


Mass: 15436.794 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Source: (natural) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A / References: UniProt: Q5HK90
#3: Protein
CRISPR system Cms endoribonuclease Csm3 / CRISPR type III A-associated RAMP protein Csm3


Mass: 24033.975 Da / Num. of mol.: 4 / Source method: isolated from a natural source
Source: (natural) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A / References: UniProt: Q5HK91
#4: 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

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RNA chain , 2 types, 2 molecules GL

#5: RNA chain RNA (37-MER)


Mass: 11811.033 Da / Num. of mol.: 1
Fragment: Staphylococcus epidermidis RP62A CRISPR RNA: Repeat plus Spacer sequence 2
Source method: isolated from a natural source
Source: (natural) Staphylococcus epidermidis RP62A (bacteria)
Strain: ATCC 35984 / RP62A
#6: RNA chain RNA (40-MER)


Mass: 12789.641 Da / Num. of mol.: 1 / Fragment: CRISPR non-self RNA target / Source method: obtained synthetically
Source: (synth.) Staphylococcus epidermidis RP62A (bacteria)

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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 effector complex with non-self target RNA
Type: COMPLEX / Entity ID: all / 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 mMNaClSodium chlorideNaClSodium chloride1
31 mMDithiothreitolC4H10O2S21
SpecimenConc.: 3.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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
Details: 3 ul of the protein-RNA complex were added to the grid and allowed to incubate for 10 seconds. After incubation the grid was blotted in a Vitrobot Mark IV (FEI Thermo Fischer) (humidity 95% ...Details: 3 ul of the protein-RNA complex were added to the grid and allowed to incubate for 10 seconds. After incubation the grid was blotted in a Vitrobot Mark IV (FEI Thermo Fischer) (humidity 95% and temperature 4 C) for 3 seconds with a force of 0 before plunge freezing in liquid ethane.

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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 FIELDBright-field microscopy / Calibrated magnification: 44454 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 49.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 2455

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
PDB_EXTRACT3.27data extraction
EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2368249
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42788 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16NBT

6nbt

A1
26NBT

6nbt

B1
36NBT

6nbt

C1
46NBT

6nbt

D1
56NBU

6nbu

J1
66NBU

6nbu

I1
76NBU

6nbu

K1
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 301.38 Å2 / Biso mean: 145.398 Å2 / Biso min: 30 Å2

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