Affinity grids in preparing single particle specimens for Electron Microscopy
Summary Immobilized metal affinity chromatography has been used extensively to isolate macromolecular complexes for structural studies. This is facilitated by the high affinity interaction between a short continuous stretch of histidine residues and nickel cations chelated by nitrilotriacetic acid (Ni-NTA). The Affinity Grid has been recently introduced as an electron microscopy (EM) grid featuring a pre-adsorbed Ni-NTA lipid-containing monolayer. Using the Affinity Grid, polyhistidine-tagged protein complexes can be prepared for single particle EM studies without prior chromatographic purification. As proof of concept, Affinity Grids were capable of purifying his-tagged transferrin-transferrin receptor complexes from concentrated Sf9 insect cell extracts when present at concentrations as low as 0.15 ?g/ml. Current procedures aimed at preparing samples for structural signal particle EM studies typically require concentrations in the 1mg/ml range. Moreover, mass spectrometry analysis has demonstrated that the purity of samples prepared via the Affinity Grid is equivalent to or in some cases superior to samples obtained by one-step Ni-affinity purification.
Applications Achieving high-throughput structure determination of macromolecular complexes remains an elusive goal of single particle EM. The principal factor limiting high throughput EM studies is sample purification. The ease of preparing samples of macromolecular complexes suitable for EM with the Affinity Grid may eliminate monolayer-mediated macromolecule purification, enabling high-throughput structure determination by single particle EM analysis.
Determining the structure of macromolecular complexes, especially of mammalian origin, by single particle EM remains a formidable challenge due to the difficulties in producing sufficient amounts of highly pure complexes that retain their structural integrity. By eliminating the need for chromatographic purification steps, even low abundance complexes become amenable to single particle EM studies. The specific binding of his-tagged complexes to the affinity Grid further concentrates the sample on the EM grid. The efficiency of the procedure is significantly improved, while concomitantly increasing the structural integrity of dynamic and unstable complexes. Importantly, the elimination of concentration steps reduces sample loss and increases the likelihood that transient protein interactions can be detected.
In addition to ease of use, monolayer-mediated purification may assist in the analysis of assembly pathways and intermediate complexes. Since the Ni-NTA monolayer adsorbs every polyhistidine-tagged complex, all intermediates are captured. Employing negative staining, images can be classified into homogenous groups for determination of assembly pathways. For Further Information Please Contact the Director of Business Development Katie Gordon Email: firstname.lastname@example.org Telephone: (617) 432-0920
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