Single-particle selection and alignment with heavy atom cluster-antibody conjugates

Abstract

A method is proposed for selecting and aligning images of single biological particles to obtain highresolution structural information by cryoelectron microscopy. The particles will be labeled with multiple heavy atom clusters to permit the precise determination of particle locations and relative orientations even when imaged close to focus with a low electron dose, conditions optimal for recording highresolution detail. Heavy atom clusters should also allow selection of images free from many kinds of defects, including specimen movement and particle inhomogeneity. Heavy atom clusters may be introduced in a general way by the construction of ''adaptor'' molecules based on single-chain Fv antibody fragments, consisting of a constant framework region engineered for optimal cluster binding and a variable antigen binding region selected for a specific target. The success of the method depends on the mobility of the heavy atom cluster on the particle, on the precision to which clusters can be located in an image, and on the sufficiency of cluster projections alone to orient and select particles for averaging. The necessary computational algorithms were developed and implemented in simulations that address the feasibility of the method.Electron microscopy of biological specimens is limited in resolution by beam-induced specimen damage because single organic molecules are destroyed by electron irradiation sufficient to reveal structural details. In addition to damaging the specimen, electrons, even at low doses, impair the quality of electron imaging by causing localized heating, specimen movement, and specimen charging. These difficulties can be overcome by image averaging and special data collection techniques (1-3). The possibility of structure determination to atomic resolution has been indicated (4) and in some cases attained (2,5,6).Image averaging to improve the signal-to-noise ratio of low-dose electron micrographs has been accomplished in the past by diffraction from ordered arrays of molecules or by computational methods of aligning the images of single particles. Development of the diffraction approach exploited naturally occurring ordered arrays, such as virus particles, muscle fibers, and two-dimensional (2-D) crystals of membrane proteins (7,8). A general method of forming 2-D crystals was devised to bring a wide range of proteins within reach of the approach (9). The necessity of forming a crystalline specimen nonetheless remains an impediment. It prevents the study of a great many biological objects, including partially irregular or inhomogeneous molecules and molecular complexes. The very large multiprotein complexes of most biological interest are especially prone to these limitations.Escape from the requirement for crystals by computational alignment of single particles relies on the detection of image details to determine the relative orientations of the particles and permit image averaging (10). The very paucity of detail in a low-dose electron micrograph that necessitates averaging unavoida...

RETRACTED: High-Affinity Gold Nanoparticle Pin to Label and Localize Histidine-Tagged Protein in Macromolecular Assemblies

“…Thus, we believe that AuNP tris-NTA should find broad application in non-covalent site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies as well as in challenging applications such as in situ electron tomography that requires protein localization in a complex and crowded milieu. We also envision other possible roles for the AuNP tris-NTA once bound to its intended target, including serving as a fiducial marker by providing high-contrast image features for particle identification in vitrified ice and aiding particle classification and/or alignment (Jensen and Kornberg, 1998). …”

An autobiographic conversation with Roger D Kornberg on his work on transcription regulation

“…Inspired by graduate student Grant Jensen, we have pursued the synthesis of large heavy atom clusters, for the purpose of structure determination without crystals at near atomic resolution. 22 This work has opened a window on a whole new realm of inorganic chemistry and materials science, through which we may pass into the future.…”

Gold nanoparticle–protein arrays improve resolution for cryo-electron microscopy

“…This site-specific single-cluster labeling of a protein is convenient to identify and localize the positions of specific chemical species or binding sites, subunits, and domains of the protein (AlBassam et al, 2002;Asturias et al, 2005;Boisset et al, 1992;Buchel et al, 2001;Datta et al, 2005;Kikkawa et al, 2000;Montesano-Roditis et al, 2001;Rappas et al, 2005;Wagenknecht et al, 1994). It has been proposed that site-specific multiple-cluster labeling of a protein could enable the precise determination of protein orientations, therefore improving resolution of single particle reconstruction (Jensen and Kornberg, 1998). Recent experimental progress to test this hypothesis has been made in labeling influenza neuraminidase using four single chain variable fragment (scFv)-Au NP conjugates (Ackerson et al, 2006).…”