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Structural Analysis of Arabidopsis thaliana Cellulose Synthase A1 Catalytic Domain
Author(s) -
Massenburg Lynnicia,
Vandavasi Venu,
O’Neill Hugh,
Kumar Manish,
Nixon Tracy
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.09237
Subject(s) - arabidopsis thaliana , monomer , cellulose , resolution (logic) , high resolution , domain (mathematical analysis) , chemistry , arabidopsis , materials science , biophysics , computer science , biochemistry , artificial intelligence , biology , mathematics , organic chemistry , polymer , gene , mathematical analysis , remote sensing , mutant , geology
Previous evidence has shown that the Arabidopsis thaliana CESA1 catalytic domain (AtCESA1catD) can form stable monomers and oligomers. Negative stain TEM and initial cryo‐EM screens of monomer revealed a fit to the cotton CESA1 computational model (Gh506). Optimized cryo‐EM screens pushed the limits of cryo‐EM sub‐100 kDal imaging to observe domain detail in the 59.7 kDal AtCESA1catD. The first cryo‐EM optimization strategy utilized the Volta Phase Plate at low defocus to capture high contrast images with more detail. This strategy yielded problematic maps and was not ideal to yield a high resolution structure. The second cryo‐EM optimization strategy used higher electron dose over a defocus range. This strategy revealed the P‐CR and CSR domains and is very promising for high resolution structures. We purified AtCESA1catD monomer and showed that negative stain structure fits to Gh506. The 2nd cryo‐EM optimization is promising for high resolution, and future work will involve picking more protein particles followed by low‐ to high‐resolution refinement. Support or Funding Information The work was fully supported as part of The Center for Ligno Cellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science. Additional support was given by the Alfred P. Sloan Foundation’s Minority Ph.D. (MPHD) Program, awarded in 2018–19 (Grant No.: G□2016‐20166039).