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Yorodumi- EMDB-6014: Dynein motor domain in the presence of ADP, with linker at position 2 -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6014 | |||||||||
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Title | Dynein motor domain in the presence of ADP, with linker at position 2 | |||||||||
Map data | Reconstruction of dynein motor domain in 100 uM ADP | |||||||||
Sample |
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Keywords | dynein / lis1 / regulation mechanism | |||||||||
Function / homology | Function and homology information karyogamy / establishment of mitotic spindle localization / astral microtubule / nuclear migration along microtubule / minus-end-directed microtubule motor activity / dynein light intermediate chain binding / cytoplasmic dynein complex / nuclear migration / spindle pole body / dynein intermediate chain binding ...karyogamy / establishment of mitotic spindle localization / astral microtubule / nuclear migration along microtubule / minus-end-directed microtubule motor activity / dynein light intermediate chain binding / cytoplasmic dynein complex / nuclear migration / spindle pole body / dynein intermediate chain binding / cytoplasmic microtubule / establishment of mitotic spindle orientation / mitotic sister chromatid segregation / cytoplasmic microtubule organization / Neutrophil degranulation / mitotic spindle organization / cell cortex / ATP hydrolysis activity / ATP binding / cytoplasm Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 19.5 Å | |||||||||
Authors | Toropova K / Zou S / Roberts AJ / Redwine WB / Goodman BS / Reck-Peterson SL / Leschziner AE | |||||||||
Citation | Journal: Elife / Year: 2014 Title: Lis1 regulates dynein by sterically blocking its mechanochemical cycle. Authors: Katerina Toropova / Sirui Zou / Anthony J Roberts / William B Redwine / Brian S Goodman / Samara L Reck-Peterson / Andres E Leschziner / Abstract: Regulation of cytoplasmic dynein's motor activity is essential for diverse eukaryotic functions, including cell division, intracellular transport, and brain development. The dynein regulator Lis1 is ...Regulation of cytoplasmic dynein's motor activity is essential for diverse eukaryotic functions, including cell division, intracellular transport, and brain development. The dynein regulator Lis1 is known to keep dynein bound to microtubules; however, how this is accomplished mechanistically remains unknown. We have used three-dimensional electron microscopy, single-molecule imaging, biochemistry, and in vivo assays to help establish this mechanism. The three-dimensional structure of the dynein-Lis1 complex shows that binding of Lis1 to dynein's AAA+ ring sterically prevents dynein's main mechanical element, the 'linker', from completing its normal conformational cycle. Single-molecule experiments show that eliminating this block by shortening the linker to a point where it can physically bypass Lis1 renders single dynein motors insensitive to regulation by Lis1. Our data reveal that Lis1 keeps dynein in a persistent microtubule-bound state by directly blocking the progression of its mechanochemical cycle. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6014.map.gz | 1.7 MB | EMDB map data format | |
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Header (meta data) | emd-6014-v30.xml emd-6014.xml | 10.7 KB 10.7 KB | Display Display | EMDB header |
Images | 400_6014.gif 80_6014.gif | 42.1 KB 3.5 KB | ||
Others | emd_6014_half_map_1.map.gz emd_6014_half_map_2.map.gz | 17 MB 17 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6014 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6014 | HTTPS FTP |
-Validation report
Summary document | emd_6014_validation.pdf.gz | 79.3 KB | Display | EMDB validaton report |
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Full document | emd_6014_full_validation.pdf.gz | 78.4 KB | Display | |
Data in XML | emd_6014_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6014 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6014 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_6014.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of dynein motor domain in 100 uM ADP | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.73 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Supplemental map: emd 6014 half map 1.map
File | emd_6014_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Supplemental map: emd 6014 half map 2.map
File | emd_6014_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Dynein motor domain in 100 uM ADP
Entire | Name: Dynein motor domain in 100 uM ADP |
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Components |
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-Supramolecule #1000: Dynein motor domain in 100 uM ADP
Supramolecule | Name: Dynein motor domain in 100 uM ADP / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 1 |
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Molecular weight | Experimental: 361 KDa / Theoretical: 361 KDa |
-Macromolecule #1: Dynein heavy chain
Macromolecule | Name: Dynein heavy chain / type: protein_or_peptide / ID: 1 / Name.synonym: DYN1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: RPY844 / synonym: Yeast / Location in cell: Cytoplasmic |
Molecular weight | Experimental: 361 KDa / Theoretical: 361 KDa |
Recombinant expression | Organism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: RPY844 |
Sequence | UniProtKB: Dynein heavy chain, cytoplasmic |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.03 mg/mL |
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Buffer | pH: 8 Details: 50 mM Tris-HCl, 150 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 1 mM DTT, 100 uM ADP |
Staining | Type: NEGATIVE Details: Grids with adsorbed protein were floated on 2% w/v uranyl formate, then sandwiched with a thin layer of carbon, blotted, and frozen in liquid nitrogen. |
Grid | Details: 200 mesh C-flat grid with thin carbon support |
Vitrification | Cryogen name: NITROGEN / Instrument: OTHER Method: Manually blot, wait 10-20 seconds, then manually plunge into liquid nitrogen. |
-Electron microscopy
Microscope | FEI TECNAI 20 |
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Date | Jan 14, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 200 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 86800 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 1.31 µm / Nominal defocus min: 0.39 µm / Nominal magnification: 62000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
CTF correction | Details: phase flip |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 19.5 Å / Resolution method: OTHER / Software - Name: RELION Details: The dataset was first 3D-classified in RELION to sort different linker positions. Particles in this map displayed a linker shifted relative to its position in the absence of nucleotide. Number images used: 3983 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera |
Details | To compare linker positions between PDB file and EM map, only coordinates for the dynein ring (AAA1-6) were fit into the EM density. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |