title: Capturing time-resolved changes in molecular structure by negative staining.
abstract: Imaging structural intermediates of biological processes is a key step in understanding biological function. Because intermediates are commonly short-lived, lasting only milliseconds, the main methods used to capture them have been conventional imaging of analog or inhibited states, having extended lifetimes, or rapid (millisecond timescale) freezing of intermediates with subsequent observation by cryo-EM. We have developed a simpler method that fixes structure on the millisecond timescale. The procedure consists of briefly (milliseconds) exposing the macromolecular structure of interest on an EM grid to conditions that initiate the structural change, then immediately fixing with uranyl acetate or tannic acid. Specimens are then observed by negative staining. The key finding that validates this approach is our demonstration that uranyl acetate, and in some cases tannic acid, fixes protein molecular structure on the millisecond timescale. This is demonstrated by our observation that exposure of actin and myosin filaments to these fixatives for as little as 10 ms is sufficient to fully preserve them against changes that normally induce rapid and major alteration in their molecular structure. Fixation appears to stabilize both ionic and hydrophobic bonds. This approach should be of general utility for studying transient molecular changes in many systems.
authors.affiliation: Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.
authors.forename: Fa-Qing
authors.initials: FQ
authors.lastname: Zhao
authors.forename: Roger
authors.initials: R
authors.lastname: Craig
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020285:Cryoelectron Microscopy / Q000379:methods*; D047348:Hydrolyzable Tannins / Q000494:pharmacology; D007477:Ions; D008274:Magnesium / Q000378:metabolism; D008853:Microscopy / Q000379:methods*; D008974:Mollusca; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D009942:Organometallic Compounds / Q000494:pharmacology; D012492:Salts / Q000378:metabolism; D013997:Time Factors
chemical_list: D000199:Actins; D047348:Hydrolyzable Tannins; D007477:Ions; D009942:Organometallic Compounds; D012492:Salts; C005460:uranyl acetate; D000255:Adenosine Triphosphate; D009218:Myosins; D008274:Magnesium; D002118:Calcium