Electron crystallography of ultrathin 3D protein crystals: Atomic model with charges

Abstract

Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca 2+ -ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca 2+ -binding sites of Ca 2+ -ATPase and that of the iron atom in the heme in catalase.electron crystallography | protein crystal | Coulomb potential | Ca 2+ -ATPase | catalase P rotein atoms scatter electrons four to five orders of magnitude more strongly than they do X-rays, thus allowing individual protein molecules to be imaged by electron microscopy (1). Although not fully exploited so far, electron protein crystallography has great potential and indeed has yielded superb high-resolution (∼2.0-Å resolution) atomic structures from 2D crystals (2). However, electron crystallography of 3D crystals is problematic, as stacking of even a few layers makes diffraction patterns discrete in all directions, and methods developed for conventional electron crystallography of 2D crystals (3) are not useful (SI Appendix, Fig. S1A, Left). This problem can be overcome, however, as Gonen and coworkers demonstrated (4, 5), by rotating the crystal to spatially integrate the intensities of diffraction spots as in X-ray crystallography (SI Appendix, Fig. S1A, Right) or, in certain cases, even combining simple tilt series.Another important feature of electron scattering is that the diffraction pattern formed by elastically scattered electrons is directly related to the distribution of Coulomb potential. This is in marked contrast to X-rays, which, because they are scattered by electrons, yield an electron density map. Coulomb potential maps may be more difficult to interpret, compared with electron density maps by X-ray crystallography, as the appearance of the same residues may differ depending on their charged state, resolution, and surrounding environment ( Fig. 1 and SI Appendix, Fig. S2), but they provide unique information, not attainable by X-rays (6). Theoretical potential maps (F calc maps; Fig. 1 B-E) calculated from an atomic model of Ca 2+ -ATPase (7) using scattering factors for 300-keV electrons highlight these features. For instance, densities of acidic residues are absent or weak when lower-resolution data are included in the map calc...

Protein structure determination by electron diffraction using a single three-dimensional nanocrystal

“…Additional hardware modifications may further benefit electron diffraction studies of macromolecular compounds, for example a reliable and well integrated goniometer tilt (Yonekura et al, 2015) and using an in-column energy filter research papers (Yonekura et al, 2015). The data presented here show that with a highly sensitive and accurate hybrid pixel detector, nanometre-sized crystals of macromolecules are now also possible targets for three-dimensional protein electron crystallography, which has the advantage of reducing the effects of dynamical diffraction.…”

“…The total diffracting volume of the crystal is no more than 6 Â 10 5 unit cells (Table 2). A comparison with structures of macromolecules previously solved by electron diffraction recorded on CCD and CMOS detectors show that these used significantly larger crystals (Nannenga, Shi, Leslie et al, 2014;Nannenga, Shi, Hattne et al, 2014;Yonekura et al, 2015;Hattne et al, 2015). Since the quality of the diffraction data from protein crystals is determined in the limiting case by the crystallinity of the sample, these data need to be interpreted with great care and should only be used to infer trends.…”

“…At higher angles, the distance that the electrons have to travel through the surrounding amorphous ice and the protein crystal can become too large for accurate data acquisition. These limitations are inherent to current implementations of electron diffraction: others have collected to up to $44 (Nannenga, Shi, Leslie et al, 2014), $61 (Nannenga, Shi, Hattne et al, 2014) and $40 (Yonekura et al, 2015). These data were collected on crystals that were significantly larger than our nanocrystals, and in case of Yonekura and coworkers needed merging from 58 and 99 crystals (Table 2).…”

“…It also then placed the second monomer. A Z-score of 22.5 is sufficiently high above the threshold of 8.0, indicating a successful structure solution (McCoy et al, 2007 Lysozyme (Nannenga, Shi, Leslie et al, 2014) 3j6k CMOS 2.5 P4 3 2 1 2 76 Â 76 Â 37 1 0.5 Â 2.0 Â 2.0 (2 mm 3 ) 9.4 18 Catalase (Nannenga, Shi, Hattne et al, 2014) 3j7b CMOS 3.2 P2 1 2 1 2 1 68 Â 172 Â 182 1 0.15 Â 4.0 Â 6.0 (3.6 mm 3 ) 1.7 14 Catalase (Yonekura et al, 2015) 3j7u CCD 3.2 P2 1 2 1 2 1 69 Â 173 Â 206 58 0.1 Â 2.0 Â 2.0 (23 mm 3 ) 9.4 77 Ca 2+ -ATPase (Yonekura et al, 2015) 3j7t CCD 3.4 C2 166 Â 64 Â 147 ( = 98 ) 99 0.1 Â 2.0 Â 2.0 (40 mm 3 ) 25 490 † The illuminated crystal size used for data acquisition is estimated from the reported crystal dimensions and the aperture sizes used; for the structures with PDB codes 3j7u and 3j7t (Yonekura et al, 2015) we assumed that the plate-like crystals had a surface area of 2 Â 2 mm. The total diffracted volume (indicated by the number in parentheses) takes the number of crystals required for the three-dimensional data set into account.…”

“…The first protein structure of a micrometre-sized crystal was determined soon after, using discrete rotation steps (Shi et al, 2013). More recently, continuous rotation became the preferred method in protein electron crystallography (Nannenga, Shi, Leslie et al, 2014;Nannenga, Shi, Hattne et al, 2014;Yonekura et al, 2015). The attractiveness of electron crystallography for macromolecular samples is further encouraged by the observation that a large fraction of seemingly failed crystallization attempts contain nanocrystals (Stevenson et al, 2014(Stevenson et al, , 2016.…”