Macromolecular diffractive imaging using imperfect crystals

Abstract

The three-dimensional structures of macromolecules and their complexes are predominantly elucidated by X-ray protein crystallography. A major limitation is access to high-quality crystals, to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields sufficiently high-resolution information that the crystal structure can be solved. The observation that crystals with shrunken unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks1,2 hints that crystallographic resolution for some macromolecules may be limited not by their heterogeneity but rather by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern, equal to the incoherent sum of diffraction from rigid single molecular complexes aligned along several discrete crystallographic orientations and hence with an increased information content3. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins4 —they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 Å limit of measurable Bragg peaks, which allows us to directly phase5 the pattern. With the molecular envelope conventionally determined at 4.5 Å as a constraint, we then obtain a static image of the photosystem II dimer at 3.5 Å resolution. This result shows that continuous diffraction can be used to overcome long-supposed resolution limits of macromolecular crystallography, with a method that puts great value in commonly encountered imperfect crystals and opens up the possibility for model-free phasing6,7.

Ultrafast electron diffraction imaging of bond breaking in di-ionized acetylene

“…Reaction imaging at the molecular level requires a combination of few-femtosecond temporal and picometer spatial measurement resolution (5). Amongst the many techniques that are currently under intense development, x-ray scattering can reach few-femtosecond pulse durations at photon energies of 8.3 keV (1.5 Å) (6) with a demonstrated measurement resolution of 3.5 Å (7). Challenges for such photonbased approaches are the coarse spatial resolution and the low scattering cross-sections, especially for gas phase investigations.…”

Ultrafast Dynamics of a Nucleobase Analogue Illuminated by a Short Intense X-ray Free Electron Laser Pulse

“…Time-resolved spectroscopy on the time scale of femtoseconds to picoseconds allows us to monitor, for the first time, atoms and electrons in action [4][5][6][7][8][9][10][11][12][13][14]. For structural analysis, the extreme intensity focused into a couple of μm 2 transforms single-shot diffraction imaging of biomolecules and nanosize objects from a remote goal into a tangible reality [15][16][17][18][19].…”

Improving the efficiency of molecular replacement by utilizing a new iterative transform phasing algorithm

“…A weighted-average electron-density map was calculated to identify a protein mask from the calculated electron-density map. The HIO method has proved to be a very effective solvent-flattening and phaserecovery technique (Liu et al, 2012;He & Su, 2015;Ayyer et al, 2016) by consistently applying constraints in real space and Fourier space (Marchesini, 2007). Like real-space phasing methods (Su, 2008), the protein mask serves as a high-density support.…”