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Yorodumi- PDB-7v01: Staphylococcus epidermidis RP62a CRISPR short effector complex wi... -
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-Basic information
Entry | Database: PDB / ID: 7v01 | |||||||||||||||
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Title | Staphylococcus epidermidis RP62a CRISPR short effector complex with self RNA target and ATP | |||||||||||||||
Components |
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Keywords | HYDROLASE/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 / metal ion binding Similarity search - Function | |||||||||||||||
Biological species | Staphylococcus epidermidis RP62A (bacteria) Staphylococcus epidermidis (bacteria) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.67 Å | |||||||||||||||
Authors | Smith, E.M. / Ferrell, S.H. / Tokars, V.L. / Mondragon, A. | |||||||||||||||
Funding support | United States, 4items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7v01.cif.gz | 421.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7v01.ent.gz | 337.8 KB | Display | PDB format |
PDBx/mmJSON format | 7v01.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7v01_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 7v01_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 7v01_validation.xml.gz | 79 KB | Display | |
Data in CIF | 7v01_validation.cif.gz | 120.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/v0/7v01 ftp://data.pdbj.org/pub/pdb/validation_reports/v0/7v01 | HTTPS FTP |
-Related structure data
Related structure data | 26925MC 7uzwC 7uzxC 7uzyC 7uzzC 7v00C 7v02C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-CRISPR system ... , 5 types, 8 molecules ABCEFHIK
#1: Protein | 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 | | 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 | | 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 | | 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 | 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 | 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 |
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#7: RNA chain | Mass: 11733.888 Da / Num. of mol.: 1 / Fragment: CRISPR self RNA target / Source method: obtained synthetically / Source: (synth.) Staphylococcus epidermidis (bacteria) |
-Non-polymers , 1 types, 2 molecules
#8: Chemical |
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-Details
Has ligand of interest | Y |
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Has protein modification | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Staphylococcus epidermidis RP62a CRISPR short effector complex with self target RNA and ATP Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES | ||||||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Staphylococcus epidermidis (bacteria) / Strain: RP62A | ||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 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 support | Details: 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 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Calibrated magnification: 46772 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 52.79 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 7527 |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1790337 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 130430 / Algorithm: FOURIER SPACE / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL | ||||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Source name: PDB / Type: experimental model
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Refinement | Cross valid method: THROUGHOUT | ||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso max: 216.49 Å2 / Biso mean: 99.426 Å2 / Biso min: 1.16 Å2 |