Abstract
Cryo electron microscopy (cryo-EM) can produce maps of macromolecules that have resolutions that are sufficiently high that structural details such as chemical modifications, water molecules and bound metal ions can be discerned. However, those accustomed to interpreting the electron-density maps of macromolecules produced by X-ray crystallography need to be careful when assigning features such as these in cryo-EM maps because cations, for example, interact far more strongly with electrons than they do with X-rays. Using simulated electrostatic potential (ESP) maps as a tool led us to re-examine a recent cryo-EM map of the human ribosome, and we realized that some of the ESP peaks originally identified as novel groups covalently bonded to the N7, O6 or O4 atoms of several guanines, adenines or uridines, respectively, in this structure are likely to instead represent Mg2+ ions coordinated to these atoms, which provide only partial charge compensation compared with Mg2+ ions located next to phosphate groups. In addition, direct evidence is provided for a variation in the level of 2'-O ribose methylation of nucleotides in the human ribosome. ESP maps can thus help in identifying ions next to nucleotide bases, i.e. at positions that can be difficult to address in cryo-EM maps due to charge effects, which are specifically encountered in cryo-EM. This work is particularly relevant to nucleoprotein complexes and shows that it is important to consider charge effects when interpreting cryo-EM maps, thus opening possibilities for localizing charges in structures that may be relevant for enzymatic mechanisms and drug interactions.
Highlights
IntroductionThe impact of high-resolution Cryo electron microscopy (cryo-EM) on structural biology has increased dramatically over the past few years thanks to the development of improved direct electron detectors and image-processing methods, which includes structure sorting by classification and methods to resolve less ordered regions by focused classification and refinement (Orlov et al, 2017; Chiu & Downing, 2017; Ognjenovicet al., 2019; von Loeffelholz et al, 2017; Klaholz, 2015; Orlova & Saibil, 2010; Khoshouei et al, 2017; Banerjee et al, 2016; Bartesaghi et al, 2015; Cheng, 2015; Nakane et al, 2018; Costa et al, 2017)
Cryo-EM maps resemble the electron-density maps generated by X-ray crystallography, and microscopists are interpreting their maps in the same way as X-ray crystallographers do (Brown et al, 2015; Natchiar et al, 2017a; Afonine, Poon et al, 2018; Afonine, Klaholz et al, 2018)
An acetyl group does fit into this density moderately well at normal contour levels, but it is clear from the simulated electrostatic potential (ESP) map (Fig. 1b), which was calculated as recently described (Wang et al, 2018; see details in Section 2), that a hydrated Mg2+ ion fits it even better at all contour levels (Figs. 1b and 1c)
Summary
The impact of high-resolution cryo-EM on structural biology has increased dramatically over the past few years thanks to the development of improved direct electron detectors and image-processing methods, which includes structure sorting by classification and methods to resolve less ordered regions by focused classification and refinement (Orlov et al, 2017; Chiu & Downing, 2017; Ognjenovicet al., 2019; von Loeffelholz et al, 2017; Klaholz, 2015; Orlova & Saibil, 2010; Khoshouei et al, 2017; Banerjee et al, 2016; Bartesaghi et al, 2015; Cheng, 2015; Nakane et al, 2018; Costa et al, 2017). Cryo-EM maps resemble the electron-density maps generated by X-ray crystallography, and microscopists are interpreting their maps in the same way as X-ray crystallographers do (Brown et al, 2015; Natchiar et al, 2017a; Afonine, Poon et al, 2018; Afonine, Klaholz et al, 2018) While this practice may be appropriate as a means for a microscopist to obtain an initial atomic model from his or her. The comparison illustrates that densities close to the N7, O6 or O4 atoms of guanosines, adenines or uridines, respectively, can be misinterpreted due to the positive charge of hydrated Mg2+ ions that appear notably larger in cryo-EM maps compared with X-ray crystallographic maps. To obtain a better understanding of these features, we have compared them with ESP maps that we have calculated for hydrated Mg2+ ions coordinately bound to these bases at the same positions
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More From: Acta Crystallographica Section D Structural Biology
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