Abstract
Cryo-EM of large, macromolecular assemblies has seen a significant increase in the numbers of high-resolution structures since the arrival of direct electron detectors. However, sub-nanometre resolution cryo-EM structures are rare compared with crystal structure depositions, particularly for relatively small particles (<400 kDa). Here we demonstrate the benefits of Volta phase plates for single-particle analysis by time-efficient cryo-EM structure determination of 257 kDa human peroxiredoxin-3 dodecamers at 4.4 Å resolution. The Volta phase plate improves the applicability of cryo-EM for small molecules and accelerates structure determination.
Highlights
Cryo-EM of large, macromolecular assemblies has seen a significant increase in the numbers of high-resolution structures since the arrival of direct electron detectors
Direct electron detectors are proving pivotal in fulfilling this promise, with the recent publications of many atomic or near-atomic models derived from Cryo-electron microscopy (cryo-EM) maps[2]
We sought to investigate the potential for structure determination using Volta potential phase plate (VPP) with single-particle analysis, for solving the cryo-EM structure of the 257-kDa, toroidal dodecamers of human peroxiredoxin-3 as an example for tackling relatively ‘small’ protein complexes
Summary
Cryo-EM of large, macromolecular assemblies has seen a significant increase in the numbers of high-resolution structures since the arrival of direct electron detectors. We demonstrate the benefits of Volta phase plates for single-particle analysis by time-efficient cryo-EM structure determination of 257 kDa human peroxiredoxin-3 dodecamers at 4.4 Å resolution. Factors that dictate the attainable resolution include particle size, rigidity, sample purity, orientation coverage and ice thickness[6] Optimizing these parameters can be complex, prohibitively time-consuming, and does not always lead to high-quality maps. The recently proposed Volta potential phase plate (VPP) addresses this long-recognized problem of cryo-EM7,8 and has many advantages over previous phase plate designs including durability and ease of use[9]. We sought to investigate the potential for structure determination using VPP with single-particle analysis, for solving the cryo-EM structure of the 257-kDa, toroidal dodecamers of human peroxiredoxin-3 We recently obtained cryo-EM reconstructions of hPrx[3] filaments and observed a structured C terminus[11], which has not been previously solved in homologous crystal structures, such as that of bovine Prx[3] (ref. 14)
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