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- EMDB-1492: hDmc1-ssDNA filament in the compressed state, determined by elect... -

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Basic information

Entry
Database: EMDB / ID: EMD-1492
TitlehDmc1-ssDNA filament in the compressed state, determined by electron microscopy and single particle analysis
Map dataThis is a 3d map of hDmc1 in the compressed state,a recombinase that plays a crucial role in faithful chromosome segregation during meiosis
Sample
  • Sample: Human Dmc1 protein complexed with ssDNA
  • Organelle or cellular component: Dmc1-ssDNA filament
KeywordsDmc1-ssDNA filament / recombinase / electron microscopy / single particle analysis
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / negative staining / Resolution: 17.0 Å
AuthorsOkorokov AL / Bugreev DV / Hodgkinson J / Mazin AV / Orlova EV
CitationJournal: PLoS One / Year: 2010
Title: Structure of the hDmc1-ssDNA filament reveals the principles of its architecture.
Authors: Andrei L Okorokov / Yuriy L Chaban / Dmitry V Bugreev / Julie Hodgkinson / Alexander V Mazin / Elena V Orlova /
Abstract: In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a ...In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination.
History
DepositionMar 4, 2008-
Header (metadata) releaseMar 5, 2008-
Map releaseMay 29, 2009-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / 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 cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_1492.jpg

Downloads & links

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Map

FileDownload / File: emd_1492.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a 3d map of hDmc1 in the compressed state,a recombinase that plays a crucial role in faithful chromosome segregation during meiosis
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.68 Å/pix.
x 160 pix.
= 268.8 Å		1.68 Å/pix.
x 160 pix.
= 268.8 Å		1.68 Å/pix.
x 160 pix.
= 268.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.68 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.475949 - 1.15959
Average (Standard dev.)0.0313314 (±0.13179)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-79-80
Dimensions160160160
Spacing160160160
CellA=B=C: 268.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.681.681.68
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z268.800268.800268.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-55-55-55
NX/NY/NZ111111111
MAP C/R/S123
start NC/NR/NS-79-80-80
NC/NR/NS160160160
D min/max/mean-0.4761.1600.031

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Supplemental data

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Sample components

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Entire : Human Dmc1 protein complexed with ssDNA

EntireName: Human Dmc1 protein complexed with ssDNA
Components
  • Sample: Human Dmc1 protein complexed with ssDNA
  • Organelle or cellular component: Dmc1-ssDNA filament

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Supramolecule #1000: Human Dmc1 protein complexed with ssDNA

SupramoleculeName: Human Dmc1 protein complexed with ssDNA / type: sample / ID: 1000 / Oligomeric state: helica filaments / Number unique components: 2
Molecular weightTheoretical: 38 KDa

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Supramolecule #1: Dmc1-ssDNA filament

SupramoleculeName: Dmc1-ssDNA filament / type: organelle_or_cellular_component / ID: 1 / Name.synonym: recombinase / Details: helical hDmc1-ssDNA nucleoprotein filament / Oligomeric state: filaments / Recombinant expression: Yes
Ref INTERPROdivclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp ...
divclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp kIPR011940 ampajax1cl asspoptrgi IPR011940i spandiv
Source (natural)Organism: Homo sapiens (human) / synonym: human / Location in cell: nucleus
Recombinant expressionOrganism: Escherichia coli EG1271 / Recombinant plasmid: pEG 8A_4

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7
Details: 25 mM Tris-acetate (pH 7.0), 2 mM ATP, 100 mM NaCl, 1 mM DTT, and 2 mM CaCl2
StainingType: NEGATIVE
Details: 2% w/v methylamine tungstate, pH 6.8 (Nano-W, Nanoprobes Inc.)
GridDetails: carbon-coated copper grids (400 mesh, freshly glow-discharged in air)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsCalibrated magnification: 41400 / Illumination mode: FLOOD BEAM / Imaging mode: OTHER / Cs: 2.4 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 44000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 1.68 µm / Number real images: 15 / Average electron dose: 25 e/Å2 / Details: densitometer, Zeiss-SCAI, a step size of 7 micron / Od range: 1.7 / Bits/pixel: 8

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Image processing

CTF correctionDetails: Each segment
Final angle assignmentDetails: Euler angles were determined by angular reconstitution and refined by projection matching
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, IMAGIC
Details: 250 classes were used for the compressed conformation, containing approximatly 15 images per class
DetailsSegments of hDmc1 filaments were selected manually

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Atomic model buiding 1

Initial modelPDB ID:

1v5w

SoftwareName: UROX and Chimera
DetailsProtocol: Rigid body. The domains were separately fitted
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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