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

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Entry
Database: EMDB / ID: EMD-4615
TitleDps-DNA crystal structure determined in vitro
Map data
SampleDps-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
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 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.
DateDeposition: Feb 15, 2019 / Header (metadata) release: May 22, 2019 / Map release: May 22, 2019 / Update: May 29, 2019

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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

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.7 Å/pix.
x 80 pix.
= 296. Å
3.7 Å/pix.
x 80 pix.
= 296. Å
3.7 Å/pix.
x 80 pix.
= 296. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.7 Å
Density
Contour LevelBy AUTHOR: 0.15 / Movie #1: 0.3
Minimum - Maximum-0.7046681 - 0.63183063
Average (Standard dev.)-0.0023388544 (±0.16253231)
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
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

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Sample components

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Entire Dps-DNA co-crystal

EntireName: Dps-DNA co-crystal
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.
Number of components: 1

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Component #1: protein, Dps-DNA co-crystal

ProteinName: Dps-DNA co-crystal
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.
Recombinant expression: No
MassTheoretical: 224 kDa
SourceSpecies: Escherichia coli BL21(DE3) (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pET-dps

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Experimental details

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Sample preparation

SpecimenSpecimen state: 3D array / Method: cryo EM
Sample solutionSpecimen conc.: 3.1 mg/mL / pH: 8
Support filmPelco EasiGlow ( 25 mA)
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 283 K / Humidity: 95 % / Details: blotting for 1.5 seconds.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.02 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 18000.0 X (nominal), 37837.0 X (calibrated) / Cs: 0.01 mm / Imaging mode: BRIGHT FIELD / Defocus: 3000.0 - 4500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON II (4k x 4k)

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Image acquisition

Image acquisitionSampling size: 14 µm

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Image processing

ProcessingMethod: subtomogram averaging / Applied symmetry: C1 (asymmetric) / Number of subtomograms: 9123 / Number of class averages: 2
3D reconstructionAlgorithm: BACK PROJECTION
CTF correction: 3D models for the contrast transfer function (CTF) in RELION2
Resolution: 13.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: Correlation coefficient / Refinement space: REAL
Input PDB model: 1DPS

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