Improving Cryo-EM Specimen Quality Using Surface Science

PROJECT SUMMARY The goal of Phase I is to adapt DeNovX’s surface science approach to improving crystallization to challenges in specimen preparation for cryogenic electron microscopy (cryoEM) that limit throughput and diminish resolution. CryoEM is superior for studies of noncrystalline biological analytes including proteins, complexes, and assemblies. Ideal cryoEM specimens require the bioanalyte to be evenly distributed and randomly oriented in vitrified aqueous media on a support. Specimen preparation comprises the deposition of aqueous bioanalyte on a support “grid”, thinning via blotting, and vitrification. Specimen quality is adversely affected by: (1) support hydrophobicity, (2) partitioning of bioanalytes to the support film surfaces, (3) partitioning of bioanalytes to the air-water interface, and (4) orientation bias. DeNovX and Vanderbilt will expand their ongoing collaboration to create innovative products using physicochemical surface modifications to tune the surface energy, hydrophilicity, H-bonding, and interfacial surface charge properties of cryoEM supports to create thermodynamically favorable domains that enhance bioanalyte incorporation while minimizing partitioning and orientation bias. Improved cryoEM workflows and specimen quality will benefit Public Health through a better structure-function understanding of diseases and potential therapies. Specific Aim 1 - Identify physical and chemical modifications to Au- and C-based cryoEM specimen supports that enhance aqueous sample adsorption across holes using replicate, controlled tensiometry and microscopy studies. Candidate physical modifications for holey supports and continuous films (graphene/graphene oxide) include surface energy tuning; texturing; etc.; and chemical modifications include plasma treatment; randomly anchored (covalent or noncovalent) combinations of polyethylene oxide (PEO), polypropylene oxide (PPO), PEO/PPO copolymers; hydroxyl-, alkyl-, or ionizable termini; etc. Confirm that the modified surfaces are comparably wettable (± 3𝜎) to control supports using solution conditions for benchmark cryoEM bioanalytes (e.g., ferritin, E. coli ribosomes, etc.). Rank and advance the top six physicochemical surface modifications showing increases of ≥ 10% in hydrophilicity/surface energy or bioanalyte adsorption across support holes. Specific Aim 2 - Produce Au- and C-based cryoEM specimen supports using the most promising physicochemical modifications from Aim 1 and conduct controlled, replicate studies (n ≥ 3) of the benchmark bioanalytes using cryoEM to demonstrate that surface modification does not decrease the S/N of vitrified specimens by ≥ 5% and that particle incorporation to holes is improved by ≥ 10% or that useful particle losses to support film or air-water partitioning are reduced by ≥ 5% relative to controls. Random particle orientations will be qualitatively assessed for challenge bioanalytes (e.g., the flagellar rotor, Krebs cycle enzymes, etc.) to confirm that orientation bias is not exacerbated . It is anticipated that more randomly oriented particles will be incorporated into cryoEM grids, and Phase II plans include maturing prototypes through technology demonstrations.