+Open data
-Basic information
Entry | Database: PDB / ID: 7pds | ||||||
---|---|---|---|---|---|---|---|
Title | The structure of PriRep1 with dsDNA | ||||||
Components |
| ||||||
Keywords | REPLICATION / Helicase / SaPI1 | ||||||
Function / homology | Virulence-associated E / Virulence-associated protein E-like domain / P-loop containing nucleoside triphosphate hydrolase / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / Similar to D. nodosus vapE Function and homology information | ||||||
Biological species | Staphylococcus aureus (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å | ||||||
Authors | Qiao, C.C. / Mir Sanchis, I. | ||||||
Funding support | Sweden, 1items
| ||||||
Citation | Journal: Nucleic Acids Res / Year: 2022 Title: Staphylococcal self-loading helicases couple the staircase mechanism with inter domain high flexibility. Authors: Cuncun Qiao / Gianluca Debiasi-Anders / Ignacio Mir-Sanchis / Abstract: Replication is a crucial cellular process. Replicative helicases unwind DNA providing the template strand to the polymerase and promoting replication fork progression. Helicases are multi-domain ...Replication is a crucial cellular process. Replicative helicases unwind DNA providing the template strand to the polymerase and promoting replication fork progression. Helicases are multi-domain proteins which use an ATPase domain to couple ATP hydrolysis with translocation, however the role that the other domains might have during translocation remains elusive. Here, we studied the unexplored self-loading helicases called Reps, present in Staphylococcus aureus pathogenicity islands (SaPIs). Our cryoEM structures of the PriRep5 from SaPI5 (3.3 Å), the Rep1 from SaPI1 (3.9 Å) and Rep1-DNA complex (3.1Å) showed that in both Reps, the C-terminal domain (CTD) undergoes two distinct movements respect the ATPase domain. We experimentally demonstrate both in vitro and in vivo that SaPI-encoded Reps need key amino acids involved in the staircase mechanism of translocation. Additionally, we demonstrate that the CTD's presence is necessary for the maintenance of full ATPase and helicase activities. We speculate that this high interdomain flexibility couples Rep's activities as initiators and as helicases. | ||||||
History |
|
-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
---|
-Downloads & links
-Download
PDBx/mmCIF format | 7pds.cif.gz | 484 KB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb7pds.ent.gz | 402.7 KB | Display | PDB format |
PDBx/mmJSON format | 7pds.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7pds_validation.pdf.gz | 1.8 MB | Display | wwPDB validaton report |
---|---|---|---|---|
Full document | 7pds_full_validation.pdf.gz | 1.8 MB | Display | |
Data in XML | 7pds_validation.xml.gz | 88.1 KB | Display | |
Data in CIF | 7pds_validation.cif.gz | 129.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/pd/7pds ftp://data.pdbj.org/pub/pdb/validation_reports/pd/7pds | HTTPS FTP |
-Related structure data
Related structure data | 13342MC 7olaC 7om0C M: map data used to model this data C: citing same article (ref.) |
---|---|
Similar structure data | Similarity search - Function & homologyF&H Search |
EM raw data | EMPIAR-10880 (Title: Staphylococcal self-loading helicases couple the staircase mechanism with inter domain high flexibility Data size: 395.0 / Data #1: PriRep1-DNA [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
|
---|---|
1 |
|
-Components
#1: Protein | Mass: 55423.895 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Staphylococcus aureus (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta / References: UniProt: Q8VLX1 #2: DNA chain | | Mass: 1512.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Staphylococcus aureus (bacteria) #3: Chemical | ChemComp-AGS / #4: Chemical | ChemComp-MG / Has ligand of interest | Y | |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: PriRep1-dsDNA / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
---|---|
Molecular weight | Value: 0.3 MDa / Experimental value: YES |
Source (natural) | Organism: Staphylococcus aureus (bacteria) / Strain: SaPI1 |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Rosetta / Plasmid: pET21a |
Buffer solution | pH: 8 |
Buffer component | Conc.: 20 mM/L / Formula: Tris |
Specimen | Conc.: 0.22 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid type: Quantifoil R2/1 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
---|---|
Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 5 sec. / Electron dose: 1.44 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 16 / Num. of real images: 3121 |
Image scans | Movie frames/image: 40 |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
EM software |
| ||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 538681 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141445 / Num. of class averages: 2 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL / Target criteria: correction coefficient | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
|