Direct isolation and imaging of viruses for cryo-electron microscopy three-dimensional reconstruction
Abstract
Affinity Grids were introduced as a rapid method of directly purifying target samples from crude extracts to electron microscopy (EM) grids. Affinity Grids employ a lipid monolayer containing functionalized nickel-nitrilotriacetic acid (Ni-NTA) lipids to purify polyhistidine-tagged complexes. The method was applied and adapted to virus-like particles, specifically T7 phage. A histidine tag was introduced at the C-terminus of the T7 phage capsid protein gene product (gp) 10, which yielded a phage particle with 415 copies of the histidine tag after assembly. The Affinity Grid was used to isolate T7 phage from Escherichia coli (E. coli) cell extracts. A mixture of particles at different structural states of T7 phage was observed, dominated by capsid I, MLD capsid II, and mature phage. The monolayer purified samples could be vitrified and allowed the cryo-EM 3D reconstructions of capsid I, MLD capsid II, and mature phage. Affinity Grids have then been used to rapidly purify Sindbis virus (SINV) from Aedes albopictus (C6/36) insect cell extracts previously dialyzed into phosphate buffered saline (PBS) by fusing a 12xHis tag to the N-terminus of the E2 glycoprotein of SINV. The resulting lipid monolayer purified samples could be vitrified as before and allowed the cryo-EM 3D reconstruction of SINV.
Degree
M.S.
Advisors
Jiang, Purdue University.
Subject Area
Virology|Biophysics
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