Experimental Methods of Structure Determination

The most powerful methods for determining the spatial structure of molecules are X-ray crystallography and NMR spectroscopy. The former requires the biomolecules to be arranged in periodic arrays in a crystal. The latter studies them in solution, usually in an isotopically labeled form. Crystals are spatially arranged in periodic arrays, and the molecules are packed by translational symmetry in three dimensions. In addition, symmetry operations such as mirror reflection, two-, three-, four-, and sixfold rotation, or inversion are applied. Crystal lattices diffract X-rays. The diffraction experiment involves a three-dimensional interference of spherical waves generated at the positions of the atoms in the lattice. Since the relative phases of the generated waves are superimposed in the different reflections and only their intensities are measured, the relative phases are not accessible by experiment. They must be regenerated by phasing methods. Only then can a Fourier transform be calculated to reveal the spatial distribution of the electron density in the crystal. The diffraction power and resolution of the crystals determine the accuracy of the resolved structure. For proteins, a resolution of 1.5-3 Å is typically achieved. The crystal structure is an average over space and time. Enhanced temperature factors give an estimate of the residual mobility of molecular moieties in a molecule. Electron microscopy is an alternative method for determining the structure of very large, often membrane-bound proteins. One either performs diffraction experiments (micro-ED method) or collects thousands of shadow projections of individual molecules in the electron beam in microscope mode (cryo-EM method). In the case of diffraction, reflection data are collected from many thousands of tiny and very thin crystals. Alternatively, the intensity of the beam can be reduced to collect larger diffraction data sets from the crystals. Cryo-EM requires the inclusion of compound samples in frozen water droplets to collect many thousands of shadow projections of individual molecules. The projection images are then assembled in the computer into an averaged 3D structure. If only small amounts of samples are available, or if crystallization fails, dissolved organic substances can be diffused into crystalline sponges and settle into large lattice cavities. NMR spectroscopy records the resonance of magnetic nuclei such as 1H, 13C, or 15N that are oriented in a strong magnetic field. Transitions between parallel and antiparallel orientations of the nuclear spins can be induced by additional fields, and the frequency at which these transitions occur depends on the chemical environment in a molecule. The spectral parameters contain information about the 3D structure of molecules in solution. Information about the spatial neighborhood of atoms, determined by the nuclear Overhauser effect (NOE), can be translated into distance maps between individual magnetic nuclei and help to determine the spatial structure of the protein. Therefore, a distance geometry approach is used in combination with molecular dynamics simulations. In many cases, the NMR structure of a protein in solution and the X-ray structure in a crystal have been shown to be in good agreement. Differences are observed for the surface-exposed residues. Protein crystals contain up to 70% water and have large water channels running through the crystal. Where appropriate, small ligand molecules can diffuse through these water-containing channels to reach binding sites on the proteins. Binding modes of small molecule ligands can be easily determined using these soaking techniques. Structural data of proteins can also be obtained with neutron radiation. In such determined structures, hydrogen atoms are revealed as strong scatterers. Protonation states of functional groups and the arrangement and dynamics of water molecules can be determined. Very strong synchrotron radiation from the X-ray free-electron laser can be used to resolve fast dynamic processes in protein crystals by serial crystallography. https://sn.pub/uhz9e7