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- EMDB-12987: Structure of Rep protein in SaPI1 -

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Basic information

Entry
Database: EMDB / ID: EMD-12987
TitleStructure of Rep protein in SaPI1
Map data
Sample
  • Organelle or cellular component: Rep1 in SaPI1
    • Protein or peptide: Rep1
Biological speciesStaphylococcus aureus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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
DepositionMay 21, 2021-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_12987.png

Downloads & links

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Map

FileDownload / File: emd_12987.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 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: 209.92 Å
α=β=γ: 90.0 °

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Supplemental data

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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
    • Protein or peptide: Rep1

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Supramolecule #1: Rep1 in SaPI1

SupramoleculeName: Rep1 in SaPI1 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria) / Strain: U93688
Molecular weightExperimental: 330 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta / Recombinant plasmid: pET28

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Macromolecule #1: Rep1

MacromoleculeName: Rep1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria) / Strain: U93688
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSHMFEMIDS RTGVLNANDW KSQLRRSATT QALKKTTTNA EIILCNDESL KGLVQYDAFE KVTKLKRLPY WRSKGDANYY WADIDTTHVI SHIDKLYNVQ FSRDLIDTVI EKEAYQNRFH PIKSMIESKS WDGIKRIETL FIDYLGAEDN HYNREVTKKW MMGAVARIYQ ...String:
GSHMFEMIDS RTGVLNANDW KSQLRRSATT QALKKTTTNA EIILCNDESL KGLVQYDAFE KVTKLKRLPY WRSKGDANYY WADIDTTHVI SHIDKLYNVQ FSRDLIDTVI EKEAYQNRFH PIKSMIESKS WDGIKRIETL FIDYLGAEDN HYNREVTKKW MMGAVARIYQ PGIKYDSMII LYGGQGVGKS TAVSKLGGHW YNQSIKTFKG DEVYKKLQGS WICEIEELSA FQKSTIEDIK GFISAIVDIY RASYGKRTER HPRQCVFVGT TNNYEFLKDQ TGNRRFFPIT TDKNKATKSP FDDLTPVVVQ QMFAEARVYF DENPTDKALL LDKEASEMAL KVQEAHSEKD ALVGEIEEFL ERPIPSDYWY RTLEEKRVSA HDVIDQDYIK LYGDGKLIEL PNAKPGAYVW RDKVCSMEIW KVMMKRDDQP QQHHLRKIDK ALRNTNYCGT VKKQTRYGEG IGKQYGFSVD LASYYKNLKV

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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 R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy #1

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
Microscopy ID1
Image recordingImage recording ID: 1 / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / 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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Electron microscopy #1~

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
Microscopy ID1
Image recordingImage recording ID: 2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number real images: 1158 / 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: 30970 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 91171
Image recording ID1

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