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- EMDB-13945: Rep-apo -

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Basic information

Entry
Database: EMDB / ID: EMD-13945
TitleRep-apo
Map data
Sample
  • Organelle or cellular component: Rep1 in SaPI1
Biological speciesStaphylococcus aureus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.96 Å
AuthorsQiao CC / Mir-Sanchis I
Funding support Sweden, 1 items
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
DepositionDec 6, 2021-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_13945.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 256 pix.
= 256. Å
1 Å/pix.
x 256 pix.
= 256. Å
1 Å/pix.
x 256 pix.
= 256. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.0109
Minimum - Maximum-0.027241597 - 0.040503714
Average (Standard dev.)0.00069395895 (±0.0022916917)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 256.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13945_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_13945_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_13945_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

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Sample components

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Entire : Rep1 in SaPI1

EntireName: Rep1 in SaPI1
Components
  • Organelle or cellular component: Rep1 in SaPI1

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Supramolecule #1: Rep1 in SaPI1

SupramoleculeName: Rep1 in SaPI1 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Staphylococcus aureus (bacteria) / Strain: U93688
Molecular weightExperimental: 330 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta / Recombinant plasmid: pET28

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chlorideSodium chloride
20.0 mMTris
0.5 mMEDTAEthylenediaminetetraacetic acid
2.0 mMDTT

Details: 20mMTris pH 8, 200 mM NaCl, 0.5mM EDTA, 2mM DTT
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf / Software - details: Zhangkai's
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationNumber classes: 4 / Avg.num./class: 30390 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionNumber classes used: 3 / Resolution.type: BY AUTHOR / Resolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 91171
FSC plot (resolution estimation)

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