+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13342 | |||||||||
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Title | The structure of PriRep1 with dsDNA | |||||||||
Map data | ||||||||||
Sample |
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Keywords | Helicase / SaPI1 / REPLICATION | |||||||||
Function / homology | Virulence-associated E / Virulence-associated protein E-like domain / P-loop containing nucleoside triphosphate hydrolase / Similar to D. nodosus vapE Function and homology information | |||||||||
Biological species | Staphylococcus aureus (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.14 Å | |||||||||
Authors | Qiao CC / Mir Sanchis I | |||||||||
Funding support | Sweden, 1 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_13342.map.gz | 166.6 MB | EMDB map data format | |
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Header (meta data) | emd-13342-v30.xml emd-13342.xml | 17.9 KB 17.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13342_fsc.xml | 12.8 KB | Display | FSC data file |
Images | emd_13342.png | 136.7 KB | ||
Masks | emd_13342_msk_1.map | 178 MB | Mask map | |
Filedesc metadata | emd-13342.cif.gz | 6.2 KB | ||
Others | emd_13342_half_map_1.map.gz emd_13342_half_map_2.map.gz | 140.7 MB 140.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13342 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13342 | HTTPS FTP |
-Validation report
Summary document | emd_13342_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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Full document | emd_13342_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | emd_13342_validation.xml.gz | 20 KB | Display | |
Data in CIF | emd_13342_validation.cif.gz | 26.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13342 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13342 | HTTPS FTP |
-Related structure data
Related structure data | 7pdsMC 7olaC 7om0C M: atomic model generated by this map C: citing same article (ref.) |
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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
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_13342.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 0.82 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_13342_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_13342_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_13342_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : PriRep1-dsDNA
Entire | Name: PriRep1-dsDNA |
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Components |
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-Supramolecule #1: PriRep1-dsDNA
Supramolecule | Name: PriRep1-dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Staphylococcus aureus (bacteria) / Strain: SaPI1 |
Molecular weight | Theoretical: 300 KDa |
-Macromolecule #1: Similar to D. nodosus vapE
Macromolecule | Name: Similar to D. nodosus vapE / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Staphylococcus aureus (bacteria) |
Molecular weight | Theoretical: 55.423895 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MFEMIDSRTG VLNANDWKSQ LRRSATTQAL KKTTTNAEII LCNDESLKGL VQYDAFEKVT KLKRLPYWRS KGDANYYWAD IDTTHVISH IDKLYNVQFS RDLIDTVIEK EAYQNRFHPI KSMIESKSWD GIKRIETLFI DYLGAEDNHY NREVTKKWMM G AVARIYQP ...String: MFEMIDSRTG VLNANDWKSQ LRRSATTQAL KKTTTNAEII LCNDESLKGL VQYDAFEKVT KLKRLPYWRS KGDANYYWAD IDTTHVISH IDKLYNVQFS RDLIDTVIEK EAYQNRFHPI KSMIESKSWD GIKRIETLFI DYLGAEDNHY NREVTKKWMM G AVARIYQP GIKYDSMIIL YGGQGVGKST AVSKLGGHWY NQSIKTFKGD EVYKKLQGSW ICEIEELSAF QKSTIEDIKG FI SAIVDIY RASYGKRTER HPRQCVFVGT TNNYEFLKDQ TGNRRFFPIT TDKNKATKSP FDDLTPVVVQ QMFAEARVYF DEN PTDKAL LLDKEASEMA LKVQEAHSEK DALVGEIEEF LERPIPSDYW YRTLEEKRVS AHDVIDQDYI KLYGDGKLIE LPNA KPGAY VWRDKVCSME IWKVMMKRDD QPQQHHLRKI DKALRNTNYC GTVKKQTRYG EGIGKQYGFS VDLASYYKNL KV UniProtKB: Similar to D. nodosus vapE |
-Macromolecule #2: polyA
Macromolecule | Name: polyA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Staphylococcus aureus (bacteria) |
Molecular weight | Theoretical: 1.512063 KDa |
Sequence | String: (DT)(DA)(DA)(DA)(DA) |
-Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 6 / Formula: AGS |
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Molecular weight | Theoretical: 523.247 Da |
Chemical component information | ChemComp-AGS: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.22 mg/mL |
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Buffer | pH: 8 / Component - Concentration: 20.0 mM/L / Component - Formula: Tris |
Grid | Model: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 16 / Number real images: 3121 / Average exposure time: 5.0 sec. / Average electron dose: 1.44 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 165000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: AB INITIO MODEL / Target criteria: correction coefficient |
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Output model | PDB-7pds: |