+Open data
-Basic information
Entry | Database: PDB / ID: 7om0 | ||||||
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Title | Structure of Primase-Helicase in SaPI5 | ||||||
Components | DNA primase | ||||||
Keywords | REPLICATION / Helicase / DNA binding / AMPPNP | ||||||
Function / homology | Function and homology information | ||||||
Biological species | Staphylococcus aureus (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||
Authors | Qiao, C.C. / Mir-Sanchis, I. | ||||||
Funding support | Sweden, 1items
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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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7om0.cif.gz | 502.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7om0.ent.gz | 403 KB | Display | PDB format |
PDBx/mmJSON format | 7om0.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/om/7om0 ftp://data.pdbj.org/pub/pdb/validation_reports/om/7om0 | HTTPS FTP |
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-Related structure data
Related structure data | 12982MC 7olaC 7pdsC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
EM raw data | EMPIAR-10881 (Title: Staphylococcal self-loading helicases couple the staircase mechanism with inter domain high flexibility Data size: 467.6 / Data #1: PriRep5-dsDNA [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 93007.031 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Staphylococcus aureus (bacteria) Gene: DQV53_04810, EQ90_04155, G6Y10_11370, HMPREF2819_07670 Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta / References: UniProt: A0A1S5ZIL8 #2: Chemical | ChemComp-ANP / Has ligand of interest | Y | |
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-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: Primase-Helicase in SaPI5 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 0.669 MDa / Experimental value: YES |
Source (natural) | Organism: Staphylococcus aureus (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 8 |
Buffer component | Conc.: 200 mM / Name: Sodium Chloride / Formula: NaCl |
Specimen | Conc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2 |
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 |
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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 / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 4 sec. / Electron dose: 1.16 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 4760 |
Image scans | Movie frames/image: 40 |
-Processing
EM software |
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CTF correction | Details: CTF correction was performed by Gctf after motion correction. Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 724786 | |||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C6 (6 fold cyclic) | |||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185358 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient |