|Entry||Database: EMDB / ID: EMD-20805|
|Title||Spastin Hexamer (unsharpened map)|
|Sample||Spastin Hexamer in complex with substrate peptide|
|Biological species||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 4.3 Å|
|Authors||Han H / Schubert HL|
|Funding support|| United States, 1 items |
|Citation||Journal: J. Biol. Chem. / Year: 2019|
Title: Structure of spastin bound to a glutamate-rich peptide implies a hand-over-hand mechanism of substrate translocation.
Authors: Han Han / Heidi L Schubert / John McCullough / Nicole Monroe / Michael D Purdy / Mark Yeager / Wesley I Sundquist / Christopher P Hill /
Abstract: Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic ...Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits. Here, we demonstrate that a human spastin binds weakly to unmodified peptides from the C-terminal segment of human tubulin a1A/B. A peptide comprising alternating glutamate and tyrosine residues binds more tightly, which is consistent with the known importance of glutamylation for spastin microtubule severing activity. A cryo-EM structure of the spastin-peptide complex at 4.2 Å resolution revealed an asymmetric hexamer in which five spastin subunits adopt a helical, spiral staircase configuration that binds the peptide within the central pore, while the sixth subunit of the hexamer is displaced from the peptide/substrate, as if transitioning from one end of the helix to the other. This configuration differs from a recently published structure of spastin from Drosophila melanogaster, which forms a six-subunit spiral without a transitioning subunit. Our structure resembles other recently reported AAA unfoldases, including the meiotic clade relative Vps4, and supports a model in which spastin utilizes a hand-over-hand mechanism of tubulin translocation and microtubule remodeling.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_20805.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.056 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Spastin Hexamer in complex with substrate peptide
|Entire||Name: Spastin Hexamer in complex with substrate peptide / Number of components: 1|
-Component #1: protein, Spastin Hexamer in complex with substrate peptide
|Protein||Name: Spastin Hexamer in complex with substrate peptide / Recombinant expression: No|
|Source||Species: Homo sapiens (human)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||pH: 8|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 57 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: FEI FALCON III (4k x 4k)|
|Processing||Method: single particle reconstruction / Number of projections: 119984|
|3D reconstruction||Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF|
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