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- EMDB-5757: Structural mechanism of the dynein powerstroke -

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Basic information

Entry
Database: EMDB / ID: EMD-5757
TitleStructural mechanism of the dynein powerstroke
Map dataReconstruction of axonemal dyneins in post-powerstroke state. In the erythro-9-[3-(2-hydroxynonyl)]-adenine inhibited sea urchin sperm flagella, the outer arm dyenins show post-powerstroke conformations.
Sample
  • Sample: Cryo-electron tomography and subtomographic average (1100 axonemal repeats) of inactive Strongylocentrotus purpuratus (sea urchin) sperm flagella.
  • Organelle or cellular component: dynein
Keywordsdynein movement / flagellar motility / axoneme
Function / homology
Function and homology information


karyogamy / nuclear migration along microtubule / astral microtubule / establishment of mitotic spindle localization / spindle pole body / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / dynein light intermediate chain binding / nuclear migration / dynein intermediate chain binding ...karyogamy / nuclear migration along microtubule / astral microtubule / establishment of mitotic spindle localization / spindle pole body / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / dynein light intermediate chain binding / nuclear migration / dynein intermediate chain binding / establishment of mitotic spindle orientation / mitotic sister chromatid segregation / cytoplasmic microtubule / cytoplasmic microtubule organization / Neutrophil degranulation / mitotic spindle organization / cell cortex / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
: / DYN1, AAA+ ATPase lid domain / : / Dynein heavy chain, ATPase lid domain / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / P-loop containing dynein motor region / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, linker / Dynein heavy chain, AAA module D4 ...: / DYN1, AAA+ ATPase lid domain / : / Dynein heavy chain, ATPase lid domain / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / P-loop containing dynein motor region / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, linker / Dynein heavy chain, AAA module D4 / Dynein heavy chain, coiled coil stalk / Dynein heavy chain / Dynein heavy chain, hydrolytic ATP-binding dynein motor region / Dynein heavy chain, ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / Dynein heavy chain AAA lid domain superfamily / Dynein heavy chain, domain 2, N-terminal / Dynein heavy chain, linker, subdomain 3 / Dynein heavy chain, AAA1 domain, small subdomain / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, N-terminal region 2 / Hydrolytic ATP binding site of dynein motor region / Microtubule-binding stalk of dynein motor / P-loop containing dynein motor region D4 / ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Dynein heavy chain, cytoplasmic
Similarity search - Component
Biological speciesStrongylocentrotus purpuratus (purple sea urchin)
Methodsubtomogram averaging / cryo EM / Resolution: 34.0 Å
AuthorsLin J / Okada K / Raytchev M / Smith MC / Nicastro D
CitationJournal: Nat Cell Biol / Year: 2014
Title: Structural mechanism of the dynein power stroke.
Authors: Jianfeng Lin / Kyoko Okada / Milen Raytchev / Maria C Smith / Daniela Nicastro /
Abstract: Dyneins are large microtubule motor proteins required for mitosis, intracellular transport and ciliary and flagellar motility. They generate force through a power-stroke mechanism, which is an ATP- ...Dyneins are large microtubule motor proteins required for mitosis, intracellular transport and ciliary and flagellar motility. They generate force through a power-stroke mechanism, which is an ATP-consuming cycle of pre- and post-power-stroke conformational changes that cause relative motion between different dynein domains. However, key structural details of dynein's force generation remain elusive. Here, using cryo-electron tomography of intact, active (that is, beating), rapidly frozen sea urchin sperm flagella, we determined the in situ three-dimensional structures of all domains of both pre- and post-power-stroke dynein, including the previously unresolved linker and stalk of pre-power-stroke dynein. Our results reveal that the rotation of the head relative to the linker is the key action in dynein movement, and that there are at least two distinct pre-power-stroke conformations: pre-I (microtubule-detached) and pre-II (microtubule-bound). We provide three-dimensional reconstructions of native dyneins in three conformational states, in situ, allowing us to propose a molecular model of the structural cycle underlying dynein movement.
History
DepositionSep 27, 2013-
Header (metadata) releaseOct 23, 2013-
Map releaseApr 23, 2014-
UpdateMay 14, 2014-
Current statusMay 14, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 126.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 126.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j67
  • Surface level: 126.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j67
  • Surface level: 126.3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j67
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5757.gif

Downloads & links

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Map

FileDownload / File: emd_5757.map.gz / Format: CCP4 / Size: 344.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of axonemal dyneins in post-powerstroke state. In the erythro-9-[3-(2-hydroxynonyl)]-adenine inhibited sea urchin sperm flagella, the outer arm dyenins show post-powerstroke conformations.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
9.86 Å/pix.
x 50 pix.
= 492.8 Å		9.86 Å/pix.
x 36 pix.
= 354.816 Å		9.86 Å/pix.
x 50 pix.
= 492.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 9.856 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 126.299999999999997 / Movie #1: 126.3
Minimum - Maximum98.742553709999996 - 152.18778992
Average (Standard dev.)122.468040470000005 (±7.22748041)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-73-32-76
Dimensions365050
Spacing365050
CellA: 492.8 Å / B: 354.816 Å / C: 492.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z9.8569.8569.856
M x/y/z503650
origin x/y/z0.0000.0000.000
length x/y/z492.800354.816492.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-32-73-76
NC/NR/NS503650
D min/max/mean98.743152.188122.468

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Supplemental data

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Sample components

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Entire : Cryo-electron tomography and subtomographic average (1100 axonema...

EntireName: Cryo-electron tomography and subtomographic average (1100 axonemal repeats) of inactive Strongylocentrotus purpuratus (sea urchin) sperm flagella.
Components
  • Sample: Cryo-electron tomography and subtomographic average (1100 axonemal repeats) of inactive Strongylocentrotus purpuratus (sea urchin) sperm flagella.
  • Organelle or cellular component: dynein

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Supramolecule #1000: Cryo-electron tomography and subtomographic average (1100 axonema...

SupramoleculeName: Cryo-electron tomography and subtomographic average (1100 axonemal repeats) of inactive Strongylocentrotus purpuratus (sea urchin) sperm flagella.
type: sample / ID: 1000
Details: The flagellar motility was completely inhibited by erythro-9-[3-(2-hydroxynonyl)]-adenine before the cryo sample preparation.
Number unique components: 1

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Supramolecule #1: dynein

SupramoleculeName: dynein / type: organelle_or_cellular_component / ID: 1
Details: Erythro-9-[3-(2-hydroxynonyl)]-adenine completely inhibited sperm flagellar motility, and kept the axonemal dyneins in post-powerstroke states.
Recombinant expression: No / Database: NCBI
Source (natural)Organism: Strongylocentrotus purpuratus (purple sea urchin) / synonym: sea urchin / Cell: sperm / Organelle: flagella

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statetissue

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Sample preparation

BufferpH: 8
Details: 360 mM NaCl, 50 mM MgCl2, 10 mM CaCl2, 10 mM KCl, 30 mM HEPES, pH 8.0, 2 mM erythro-9-[3-(2-hydroxynonyl)]-adenine
GridDetails: Quantifoil holey carbon grids Cu 200 mesh R2/2
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER / Method: Blot for 1.5-2.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureAverage: 80 K
Specialist opticsEnergy filter - Name: GATAN postcolumn filter GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateApr 28, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (2k x 2k) / Average electron dose: 100 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 8.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 13500
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -65 ° / Tilt series - Axis1 - Max angle: 65 °
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 34.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD, PEET
Details: Final maps were calculated by averaging 1100 particles from 9 tomograms. 1100 axonemal repeats (96 nm long) from 9 tomograms (reconstructed using fiducial alignment and weighted ...Details: Final maps were calculated by averaging 1100 particles from 9 tomograms. 1100 axonemal repeats (96 nm long) from 9 tomograms (reconstructed using fiducial alignment and weighted backprojection, IMOD software, Kremer et al. 1996) were aligned and averaged using the PEET software (bio3d.colorado.edu, Nicastro et al. 2006).
Number subtomograms used: 1100

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