Sample Preparation Induced Artifacts in Cryo-Electron Tomographs

Plevka, Pavel; Battisti, Anthony J.; Winkler, Dennis C.; Tars, K.; Holdaway, Heather A.; Bator, Carol M.; Rossmann, Michael G.

doi:10.1017/s1431927612001298

Cited by 3 publications

(3 citation statements)

References 22 publications

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“…Confusion occurred when the trial icosahedral symmetry axes were oriented with one of the icosahedral 2-fold axes parallel to an orthorhombic crystallographic 2-fold axis, resulting in a large but erroneous, locked rotation function peak. This was avoided (Plevka et al 2012 a ) by testing only the 3- and 5-fold NCS axes when exploring different orientations of the set of icosahedral axes in a locked rotation function search (Rossmann et al 1972; Tong & Rossmann, 1990).…”

Section: Small Icosahedral Non-enveloped Viruses: the Jelly-roll Foldmentioning

confidence: 99%

Structure of viruses: a short history

Rossmann

2013

Quart. Rev. Biophys.

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This review is a partially personal account of the discovery of virus structure and its implication for virus function. Although I have endeavored to cover all aspects of structural virology and to acknowledge relevant individuals, I know that I have favored taking examples from my own experience in telling this story. I am anxious to apologize to all those who I might have unintentionally offended by omitting their work. The first knowledge of virus structure was a result of Stanley's studies of tobacco mosaic virus (TMV) and the subsequent X-ray fiber diffraction analysis by Bernal and Fankuchen in the 1930s. At about the same time it became apparent that crystals of small RNA plant and animal viruses could diffract X-rays, demonstrating that viruses must have distinct and unique structures. More advances were made in the 1950s with the realization by Watson and Crick that viruses might have icosahedral symmetry. With the improvement of experimental and computational techniques in the 1970s, it became possible to determine the three-dimensional, near-atomic resolution structures of some small icosahedral plant and animal RNA viruses. It was a great surprise that the protecting capsids of the first virus structures to be determined had the same architecture. The capsid proteins of these viruses all had a 'jelly-roll' fold and, furthermore, the organization of the capsid protein in the virus were similar, suggesting a common ancestral virus from which many of today's viruses have evolved. By this time a more detailed structure of TMV had also been established, but both the architecture and capsid protein fold were quite different to that of the icosahedral viruses. The small icosahedral RNA virus structures were also informative of how and where cellular receptors, anti-viral compounds, and neutralizing antibodies bound to these viruses. However, larger lipid membrane enveloped viruses did not form sufficiently ordered crystals to obtain good X-ray diffraction. Starting in the 1990s, these enveloped viruses were studied by combining cryo-electron microscopy of the whole virus with X-ray crystallography of their protein components. These structures gave information on virus assembly, virus neutralization by antibodies, and virus fusion with and entry into the host cell. The same techniques were also employed in the study of complex bacteriophages that were too large to crystallize. Nevertheless, there still remained many pleomorphic, highly pathogenic viruses that lacked the icosahedral symmetry and homogeneity that had made the earlier structural investigations possible. Currently some of these viruses are starting to be studied by combining X-ray crystallography with cryo-electron tomography.

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Section: Small Icosahedral Non-enveloped Viruses: the Jelly-roll Foldmentioning

confidence: 99%

Structure of viruses: a short history

Rossmann

2013

Quart. Rev. Biophys.

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“…However, the absorption of proteins on graphene is complicated and protein dependent. In addition, the interface between the film and water may cause different problems (Plevka et al, 2012). Collection of tilted images has also been used to compensate for preferred orientation (Tan et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Effect of charge on protein preferred orientation at the air–water interface in cryo-electron microscopy

Zhu

Shi

et al. 2021

Journal of Structural Biology

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Section: Introductionmentioning

confidence: 99%

Effect of charge on protein preferred orientation at the air–water interface in cryo-electron microscopy

Zhu

Shi

et al. 2021

Preprint

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The air-water interface (AWI) tends to absorb proteins and frequently causes preferred orientation problems in cryo-electron microscopy (cryo-EM). Here, we examined cryo-EM data from protein samples frozen with different detergents and found that both anionic and cationic detergents promoted binding of proteins to the AWI. By contrast, nonionic and zwitterionic detergents tended to prevent proteins from attaching to the AWI. This ability was positively associated with the critical micelle concentration of the detergent. The protein orientation distributions with different anionic detergents were similar and resembled that obtained without detergent. By contrast, cationic detergents gave distinct orientation distributions. The AWI is negatively charged and the absorption of cationic detergents to the AWI alters its charge. Our results indicates that proteins absorb to charged interface and the negative charge of the AWI plays an important role in absorbing proteins in the conventional cryo-EM sample preparation. According to these findings, a new method was developed to modify the charge distribution of the AWI by adding a very low concentration of anionic detergent. Using this method, the protein particles exhibited a more evenly distributed orientations and still absorbed to the AWI enabling them embedding in a thin layer of ice, which will benefit the cryo-EM structural determination.

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Sample Preparation Induced Artifacts in Cryo-Electron Tomographs

Cited by 3 publications

References 22 publications

Structure of viruses: a short history

Structure of viruses: a short history

Effect of charge on protein preferred orientation at the air–water interface in cryo-electron microscopy

Effect of charge on protein preferred orientation at the air–water interface in cryo-electron microscopy

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