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- EMDB-4615: Dps-DNA crystal structure determined in vitro -

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Basic information

Entry
Database: EMDB / ID: EMD-4615
TitleDps-DNA crystal structure determined in vitro
Map data
Sample
  • Complex: Dps-DNA co-crystal
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 13.5 Å
AuthorsChesnokov YM / Kamyshinsky RA / Orekhov AS / Vasiliev AL
Funding support Russian Federation, 1 items
OrganizationGrant numberCountry
Russian Science Foundation18-74-10071 Russian Federation
CitationJournal: FEBS Lett / Year: 2019
Title: Protective Dps-DNA co-crystallization in stressed cells: an in vitro structural study by small-angle X-ray scattering and cryo-electron tomography.
Authors: Liubov A Dadinova / Yurii M Chesnokov / Roman A Kamyshinsky / Ivan A Orlov / Maxim V Petoukhov / Andrey A Mozhaev / Ekaterina Yu Soshinskaya / Vassili N Lazarev / Valentin A Manuvera / Anton ...Authors: Liubov A Dadinova / Yurii M Chesnokov / Roman A Kamyshinsky / Ivan A Orlov / Maxim V Petoukhov / Andrey A Mozhaev / Ekaterina Yu Soshinskaya / Vassili N Lazarev / Valentin A Manuvera / Anton S Orekhov / Alexander L Vasiliev / Eleonora V Shtykova /
Abstract: Under severe or prolonged stress, bacteria produce a nonspecific DNA-binding protein (Dps), which effectively protects DNA against damaging agents both in vitro and in vivo by forming intracellular ...Under severe or prolonged stress, bacteria produce a nonspecific DNA-binding protein (Dps), which effectively protects DNA against damaging agents both in vitro and in vivo by forming intracellular biocrystals. The phenomenon of protective crystallization of DNA in living cells has been intensively investigated during the last two decades; however, the results of studies are somewhat contradictory, and up to now, there has been no direct determination of a Dps-DNA crystal structure. Here, we report the in vitro analysis of the vital process of Dps-DNA co-crystallization using two complementary structural methods: synchrotron small-angle X-ray scattering in solution and cryo-electron tomography. Importantly, for the first time, the DNA in the co-crystals was visualized, and the lattice parameters of the crystalline Dps-DNA complex were determined.
History
DepositionFeb 15, 2019-
Header (metadata) releaseMay 22, 2019-
Map releaseMay 22, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4615.png

Downloads & links

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Map

FileDownload / File: emd_4615.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 3.7 Å
Density
Contour LevelBy AUTHOR: 0.15 / Movie #1: 0.3
Minimum - Maximum-2.0031955 - 1.8858155
Average (Standard dev.)0.001427492 (±0.4656309)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 296.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.73.73.7
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z296.000296.000296.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-2.0031.8860.001

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Supplemental data

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Mask #1

Fileemd_4615_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_4615_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_4615_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dps-DNA co-crystal

EntireName: Dps-DNA co-crystal
Components
  • Complex: Dps-DNA co-crystal

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Supramolecule #1: Dps-DNA co-crystal

SupramoleculeName: Dps-DNA co-crystal / type: complex / ID: 1 / Parent: 0
Details: Dps protein and DNA were mixed in the concentrations corresponding to the Dps/DNA ratio 1Dps dodecamer/60 bp of DNA. A ring vector pcDNA-hIRR-GFP 9900 bp was used as DNA sample.
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET-dps
Molecular weightTheoretical: 224 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state3D array

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Sample preparation

Concentration3.1 mg/mL
BufferpH: 8
Component:
ConcentrationFormula
50.0 mMTris-HCl
50.0 mMNaCl
0.5 mMEDTA
GridMaterial: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026 kPa / Details: Pelco EasiGlow ( 25 mA)
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: blotting for 1.5 seconds.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Cs image corrector (CEOS, Germany)
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 1.02 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 37837 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 18000
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

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 13.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 9123
ExtractionNumber tomograms: 11 / Number images used: 40286 / Reference model: 1DPS / Method: template matching
CTF correctionDetails: 3D models for the contrast transfer function (CTF) in RELION2
Final 3D classificationNumber classes: 2 / Avg.num./class: 20143
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1dps

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient

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