EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND
664726
Germany
German Research Foundation (DFG)
GA 2519/1-2
Germany
German Research Foundation (DFG)
RA 1781/4-2
Germany
Citation
Journal: Biol Chem / Year: 2023 Title: Asymmetric horseshoe-like assembly of peroxisomal yeast oxalyl-CoA synthetase. Authors: Jérôme Bürgi / Pascal Lill / Evdokia-Anastasia Giannopoulou / Cy M Jeffries / Grzegorz Chojnowski / Stefan Raunser / Christos Gatsogiannis / Matthias Wilmanns / Abstract: Oxalyl-CoA synthetase from is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting ...Oxalyl-CoA synthetase from is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however is partly dispensable, suggesting additional receptor bindings sites. To unravel any additional features that may contribute to its capacity to be recognized as peroxisomal target, we determined its assembly and overall architecture by an integrated structural biology approach, including X-ray crystallography, single particle cryo-electron microscopy and small angle X-ray scattering. Surprisingly, it assembles into mixture of concentration-dependent dimers, tetramers and hexamers by dimer self-association. Hexameric particles form an unprecedented asymmetric horseshoe-like arrangement, which considerably differs from symmetric hexameric assembly found in many other protein structures. A single mutation within the self-association interface is sufficient to abolish any higher-level oligomerization, resulting in a homogenous dimeric assembly. The small C-terminal domain of yeast Oxalyl-CoA synthetase is connected by a partly flexible hinge with the large N-terminal domain, which provides the sole basis for oligomeric assembly. Our data provide a basis to mechanistically study peroxisomal translocation of this target.
Model: Quantifoil / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
Vitrification
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298.15 K / Instrument: GATAN CRYOPLUNGE 3 Details: 90% humidity in the sample chamber. Gentle Blot function, 2.5 seconds.
-
Electron microscopy
Microscope
TFS KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum
Image recording
Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-60 / Number grids imaged: 4 / Number real images: 5594 / Average electron dose: 63.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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