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- EMDB-6462: Subvolumes of individual DNA minicircles showing various shapes o... -

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Basic information

Entry
Database: EMDB / ID: EMD-6462
TitleSubvolumes of individual DNA minicircles showing various shapes of the minicircles
Map dataSubtomogram of a DNA minicircle
Sample
  • Sample: Individual DNA minicircles of various shapes
  • DNA: Minicircle DNA
KeywordsDNA minicircle / cryo-ET / subtomogram / supercoiled DNA / DNA minicircle shapes
Biological speciesunidentified (others)
Methodelectron tomography / cryo EM
AuthorsIrobalieva RN / Fogg JM / Catanese DJ / Sutthibutpong T / Chen M / Barker AK / Ludtke SJ / Harris SA / Schmid MF / Chiu W / Zechiedrich L
CitationJournal: Nat Commun / Year: 2015
Title: Structural diversity of supercoiled DNA.
Authors: Rossitza N Irobalieva / Jonathan M Fogg / Daniel J Catanese / Thana Sutthibutpong / Muyuan Chen / Anna K Barker / Steven J Ludtke / Sarah A Harris / Michael F Schmid / Wah Chiu / Lynn Zechiedrich /
Abstract: By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) ...By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.
History
DepositionSep 9, 2015-
Header (metadata) releaseOct 7, 2015-
Map releaseOct 21, 2015-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6462.gif

Downloads & links

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Map

FileDownload / File: emd_6462.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram of a DNA minicircle
Voxel sizeX=Y=Z: 4.52 Å
Density
Contour LevelBy EMDB: 0.19 / Movie #1: 0.2
Minimum - Maximum-0.42069978 - 0.56056619
Average (Standard dev.)-0.00000356 (±0.07413493)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 723.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.524.524.52
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z723.200723.200723.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-8211244
NX/NY/NZ115138149
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-0.4210.561-0.000

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Supplemental data

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Supplemental map: fig-8.map

Filefig-8.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: needle 1.map

Fileneedle_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: open-8.map

Fileopen-8.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: racquet.map

Fileracquet.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 : Individual DNA minicircles of various shapes

EntireName: Individual DNA minicircles of various shapes
Components
  • Sample: Individual DNA minicircles of various shapes
  • DNA: Minicircle DNA

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Supramolecule #1000: Individual DNA minicircles of various shapes

SupramoleculeName: Individual DNA minicircles of various shapes / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 220 KDa / Method: average MW of DNA bases

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Macromolecule #1: Minicircle DNA

MacromoleculeName: Minicircle DNA / type: dna / ID: 1 / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 220 KDa
SequenceString: TTTATACTAA CTTGAGCGAA ACGGGAAGGG TTTTCACCGA TATCACCGAA ACGCGCGAGG CAGCTGTATG GCATGAAAGA GTTCTTCCCG GAAAACGCGG TGGAATATTT CGTTTCCTAC TACGACTACT ATCAGCCGGA AGCCTATGTA CCGAGTTCCG ACACTTTCAT ...String:
TTTATACTAA CTTGAGCGAA ACGGGAAGGG TTTTCACCGA TATCACCGAA ACGCGCGAGG CAGCTGTATG GCATGAAAGA GTTCTTCCCG GAAAACGCGG TGGAATATTT CGTTTCCTAC TACGACTACT ATCAGCCGGA AGCCTATGTA CCGAGTTCCG ACACTTTCAT TGAGAAAGAT GCCTCAGCTC TGTTACAGGT CACTAATACC ATCTAAGTAG TTGATTCATA GTGACTGCAT ATGTTGTGTT TTACAGTATT ATGTAGTCTG TTTTTTATGC AAAATCTAAT TTAATATATT GATATTTATA TCATTTTACG TTTCTCGTTC AGCTTT

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 8.3 / Details: 40 mM Tris-acetate, 10 mM CaCl2
GridDetails: 200 mesh holey carbon grids, glow-discharged for 25 seconds
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 95 K / Instrument: FEI VITROBOT MARK IV / Method: 1 blot, 2 seconds

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Electron microscopy

MicroscopeJEOL 2200FS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 25000
Specialist opticsEnergy filter - Name: In-column omega energy filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 25.0 eV
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 3 °
DateApr 19, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 100 e/Å2

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Image processing

Final reconstructionNumber images used: 41

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