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- PDB-7pds: The structure of PriRep1 with dsDNA -

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Basic information

Entry
Database: PDB / ID: 7pds
TitleThe structure of PriRep1 with dsDNA
Components
  • Similar to D. nodosus vapE
  • polyA
KeywordsREPLICATION / Helicase / SaPI1
Function / homologyVirulence-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 speciesStaphylococcus aureus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsQiao, C.C. / Mir Sanchis, I.
Funding support Sweden, 1items
OrganizationGrant numberCountry
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: 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
DepositionAug 6, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 10, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Aug 24, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update
Revision 1.4Nov 13, 2024Group: Data collection / Structure summary / Category: em_admin / pdbx_entry_details
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Similar to D. nodosus vapE
B: Similar to D. nodosus vapE
C: Similar to D. nodosus vapE
D: Similar to D. nodosus vapE
E: Similar to D. nodosus vapE
F: Similar to D. nodosus vapE
M: polyA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)337,29217
Polymers334,0557
Non-polymers3,23710
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37580 Å2
ΔGint-85 kcal/mol
Surface area108520 Å2

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Components

#1: Protein
Similar to D. nodosus vapE


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 polyA


Mass: 1512.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Staphylococcus aureus (bacteria)
#3: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: PriRep1-dsDNA / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 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 solutionpH: 8
Buffer componentConc.: 20 mM/L / Formula: Tris
SpecimenConc.: 0.22 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPU2.7.1image acquisition
4GctfCTF correctionZhangkai's
7Coot0.9.4.1model fitting
9RELION3.1.1initial Euler assignment
11RELION3.1.1classification
12RELION3.1.13D reconstruction
13PHENIX1.19.1-4122-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 538681
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141445 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: correction coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00423068
ELECTRON MICROSCOPYf_angle_d0.47431160
ELECTRON MICROSCOPYf_dihedral_angle_d11.8943099
ELECTRON MICROSCOPYf_chiral_restr0.0393307
ELECTRON MICROSCOPYf_plane_restr0.0043910

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