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- EMDB-13342: The structure of PriRep1 with dsDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-13342
TitleThe structure of PriRep1 with dsDNA
Map data
Sample
  • Complex: PriRep1-dsDNA
    • Protein or peptide: Similar to D. nodosus vapE
    • DNA: polyA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsHelicase / SaPI1 / REPLICATION
Function / homologyVirulence-associated E / Virulence-associated protein E-like domain / P-loop containing nucleoside triphosphate hydrolase / Similar to D. nodosus vapE
Function and homology information
Biological speciesStaphylococcus aureus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.14 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
DepositionAug 6, 2021-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13342.png

Downloads & links

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Map

FileDownload / File: emd_13342.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 360 pix.
= 295.2 Å		0.82 Å/pix.
x 360 pix.
= 295.2 Å		0.82 Å/pix.
x 360 pix.
= 295.2 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0192
Minimum - Maximum-0.049733385 - 0.10804147
Average (Standard dev.)0.0001557462 (±0.0035764782)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 295.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13342_msk_1.map
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Half map: #2

Fileemd_13342_half_map_1.map
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Half map: #1

Fileemd_13342_half_map_2.map
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Sample components

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Entire : PriRep1-dsDNA

EntireName: PriRep1-dsDNA
Components
  • Complex: PriRep1-dsDNA
    • Protein or peptide: Similar to D. nodosus vapE
    • DNA: polyA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: PriRep1-dsDNA

SupramoleculeName: PriRep1-dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Staphylococcus aureus (bacteria) / Strain: SaPI1
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: Similar to D. nodosus vapE

MacromoleculeName: Similar to D. nodosus vapE / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 55.423895 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Macromolecule #2: polyA

MacromoleculeName: polyA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 1.512063 KDa
SequenceString:
(DT)(DA)(DA)(DA)(DA)

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Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 6 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.22 mg/mL
BufferpH: 8 / Component - Concentration: 20.0 mM/L / Component - Formula: Tris
GridModel: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 538681
Startup modelType of model: NONE
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.1) / Number images used: 141445
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1.1)
Final angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.1.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: correction coefficient
Output model

PDB-7pds:
The structure of PriRep1 with dsDNA

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