+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13945 | |||||||||
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Title | Rep-apo | |||||||||
Map data | ||||||||||
Sample |
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Biological species | Staphylococcus aureus (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.96 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_13945.map.gz | 58.8 MB | EMDB map data format | |
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Header (meta data) | emd-13945-v30.xml emd-13945.xml | 16.1 KB 16.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13945_fsc.xml | 9.2 KB | Display | FSC data file |
Images | emd_13945.png | 91.3 KB | ||
Masks | emd_13945_msk_1.map | 64 MB | Mask map | |
Others | emd_13945_half_map_1.map.gz emd_13945_half_map_2.map.gz | 48.2 MB 48.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13945 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13945 | HTTPS FTP |
-Validation report
Summary document | emd_13945_validation.pdf.gz | 790.2 KB | Display | EMDB validaton report |
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Full document | emd_13945_full_validation.pdf.gz | 789.7 KB | Display | |
Data in XML | emd_13945_validation.xml.gz | 15.3 KB | Display | |
Data in CIF | emd_13945_validation.cif.gz | 19.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13945 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13945 | HTTPS FTP |
-Related structure data
Related structure data | 7olaC 7om0C 7pdsC C: citing same article (ref.) |
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EM raw data | EMPIAR-10945 (Title: Staphylococcal self-loading helicases couple the staircase mechanism 1 with inter domain high flexibility Data size: 1.6 TB / Data #1: Rep1apo [micrographs - multiframe]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_13945.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_13945_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_13945_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_13945_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Rep1 in SaPI1
Entire | Name: Rep1 in SaPI1 |
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Components |
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-Supramolecule #1: Rep1 in SaPI1
Supramolecule | Name: Rep1 in SaPI1 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Staphylococcus aureus (bacteria) / Strain: U93688 |
Molecular weight | Experimental: 330 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: Rosetta / Recombinant plasmid: pET28 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.2 mg/mL | |||||||||||||||
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Buffer | pH: 8 Component:
Details: 20mMTris pH 8, 200 mM NaCl, 0.5mM EDTA, 2mM DTT | |||||||||||||||
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Pressure: 37.0 kPa | |||||||||||||||
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 / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-40 / Number real images: 3345 / Average exposure time: 4.0 sec. / Average electron dose: 1.08 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 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 |