<p>Cryogenic
transmission electron microscopy (cryoEM) has become an increasingly common
tool for determining structures of proteins and protein complex at near atomic
resolution. We seek to determine the structure of p97 by cryoEM using an
affinity capture approach that employs a family of novel synthetic lipids
bearing water soluble PEG units and known high affinity inhibitor molecules at
the distal end of the polymer. A library of inhibitor modified affinity lipopolymers
of 5000 KD PEG molecular weights were synthesized. The inhibitor modified lipid
coated grids were used to capture p97.
The reconstruction of p97 revealed the structure at dimeric state at 3.64 Å and
monomeric state at 4.33 Å. A PEG unit
composed of 20000 KD molecular weight based polyrotaxane containing NTA ligand
as affinity tag has been synthesized, used to concentrate 6x-his tagged p97 on
TEM which also enabled to see all 3D orientation of the target particles and an
initial model of 10.64 Å resolution of p97 structure was
resolved. </p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/8020598 |
| Date | 12 October 2021 |
| Creators | Md R Hoq (6617981) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/DEVELOPMENT_OF_AFFINITY_GRID_MATERIALS_FOR_CRYOELCTRONIC_MICROSCOPY/8020598 |
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