Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Utilizing Cryo-Electron Microscopy to Characterize Proteins Relevant to Biomass Biosynthesis and Bioconversion


Samantha Ziegler1,5* (, Brandon Knott1,5, Josephine Gruber1,5, Vivek Bharadwaj1,5, Samantha Hennen1,5, Pradeep Prabhakar2,5, Lydia Joubert3, Trevor Moser4, Breeanna Urbanowicz2,5, Yannick Bomble1,5, Gerald A. Tuskan5


1Bioenergy, Science, and Technology Directorate, National Renewable Energy Laboratory; 2University of Georgia–Athens; 3Stanford-SLAC Cryo-EM Center; 4Environmental Molecular Sciences Division, Pacific Northwest National Laboratory; 5Center for Bioenergy Innovation, Oak Ridge National Laboratory



The Center for Bioenergy Innovation (CBI) vision is to accelerate domestication of bioenergy-relevant, non-model plants and microbes to enable high-impact innovations along the bioenergy and bioproduct supply chain while focusing on sustainable aviation fuels (SAF). CBI has four overarching innovation targets: (1) develop sustainable, process-advantaged biomass feedstocks; (2) refine consolidated bioprocessing with cotreatment to create fermentation intermediates; (3) advance lignin valorization for bio-based products and aviation fuel feedstocks; and (4) improve catalytic upgrading for SAF blendstocks certification.


The degradation of plant material via bacterial digestion to be converted into bioproducts such as ethanol is a main CBI focus. Plant cell walls are comprised of cellulose, hemicellulose, and lignin, which combine to make the cell wall recalcitrant to total digestion. Historically, cryo-electron microscopy (cryo-EM) was essential to confirming the structure of the cellulose synthase rosette. Solving these problems requires deeper understanding of both the biosynthesis pathways to make recalcitrant biomass polymers and the conversion pathways to break down biomass. Here, the research team utilized cryo-EM to further delve into key enzymes and complexes, with the aim of understanding biomass biosynthesis and bioconversion at a molecular level.

The first target of this work is to investigate the poorly explored proteins necessary for hemicellulose formation, which has a complex branching pattern. Most hemicellulose synthesis occurs in the Golgi apparatus in a non-templated manner, meaning that the branching sugar chains are added to the main xylan backbone presumably due to local protein interaction networks (Chou et al. 2015). By examining the near-atomic structures of glycosyltransferase proteins as determined by cryo-EM, both alone and in complex, the molecular mechanisms of hemicellulose synthesis and branching can be determined. The aim is to modulate the pathways to create less recalcitrant plants that remain robust and grow rapidly (Smith et al. 2022).

Another target for plant recalcitrance is to examine the digesting bacterium. Clostridium thermocellum is one of the best bioprocessing organisms identified to date. However, it is hampered in its ability to produce industrially relevant titers of bioproducts, such as ethanol. In past directed evolution studies regarding ethanol tolerance in C. thermocellum, one of the most frequently mutated proteins was AdhE, an alcohol-aldehyde dehydrogenase that produces ethanol. AdhE forms fascinating, spring-like ultrastructures that contain up to one hundred AdhE monomers. Using cryo-EM, the research team solved the highest resolution structure of the AdhE ultrastructure to date, providing insight into the protein’s catalytic pockets, as well as furthering understanding of intermediate aldehyde channeling (Ziegler et al. 2024). The results from the cryo-EM structure are feeding directly into mutagenesis studies to increase C. thermocellum ethanol production and tolerance.


Chou, Y-H., et al. 2015. “Protein–Protein Interactions Among Xyloglucan-Synthesizing Enzymes and Formation of Golgi-Localized Multiprotein Complexes,” Plant and Cell Physiology 56(2), 255–67.

Smith, P. J., et al. 2022. “Enzymatic Synthesis of Xylan Microparticles with Tunable Morphologies,” ACS Materials Au 2(4), 440–52.

Ziegler, S. J., et al. 2024. “Structural Characterization and Dynamics of AdhE Ultrastructures from Clostridium thermocellum: A Containment Strategy for Toxic Intermediates?” bioRxiv. DOI:10.1101/2024.02.16.580662.

Funding Information

Funding was provided by CBI led by Oak Ridge National Laboratory (ORNL). CBI is funded as a DOE Bioenergy Research Center supported by the BER Program in the DOE Office of Science under FWP ERKP886. ORNL is managed by UT-Battelle, LLC, for DOE under contract no. DE-AC05-00OR22725.