X-Ray Crystallography: Producing Crystals Feb 17-19, 2014 X-Ray Crystallography • Method to determine the ATOMIC structure of a molecule • The most widely used technique for determine structures of biological molecules • Can also be used to determine small molecules • Most entries in the PDB were determined by X-ray crystallography • NMR depends on interactions between nuclei X-Rays interacts with electrons. X-Ray • Why X-rays?? • X-rays are part of the EM spectrum • Atomic resolution - the position of two bonded atoms can be distinguished • Typical covalent bond is about 0.12 nm or 1.2x10-10m • 1x10-10 m is called an Angstrom (Å) • Limit of resolution of any optical method is defined by half the wavelength of the incident radiation • This is a consequence of the wave like properties of light • wavelength of visible light (400 to 800nm) therefore it can resolve objects to about 200nm (many cellular organelles) • For atomic resolution 0.12nm x2=0.24nm • This falls into the range of X-rays. • NO LENS for X-rays Light Microscope vs X-Ray Diffraction Light Microscopy X-ray Diffraction Sample X-rays X-ray source There is no lens for X-rays Crystals • X-rays weakly interact with matter • Crystals serve two purposes • High concentration • Order • Crystals are solids that are exact repeats • Comparison of crystalline quartz and glass - same molecule but quartz is an ordered crystal and glass is amorphous solid • Crystals are composed of repeating units - translational and rotational symmetry operators relate molecules to each other • Biological molecules are composed of chiral molecules. • Chirality limits the types of symmetry that can be used to describe a crystal • No mirror planes or points of symmetry Growing Crystals • There is no straightforward way to grow crystals • Protein crystals are a solid but do contain a lot of solvent - Most have 30-70% solvent • Large amounts of purified material • Empirical process that requires careful observation Growing Crystals II • Molecules come out of solution when its concentration exceeds it intrinsic solubility • Intrinsic solubility is related to the properties of the molecule (ie basic, acidic, hydrophobic, etc) and conditions (ie salt concentrations, pH, temperature) • Make a supersaturated solution and slowly precipitate the molecule in an orderly fashion • Methods • Remove excess solvent • This method is done for many small molecules • Not so useful for proteins since there may be other additives (salt, detergent, etc) • Decrease the solubility of molecule • This is accomplished by increasing or decreasing ionic strength of the solution or the addition of a molecule to dehydrate the protein (PEG) Crystal Growth in Theory Crystal Growth in Practice Past and Present Technology Definitions •Unit Cell - Repeating unit that builds the crystal •Asymmetric unit - the smallest portion of a crystal structure to which symmetry operations can be applied in order to generate the complete unit cell •A crystal asymmetric unit may contain •one biological assembly •a portion of a biological assembly •multiple biological assemblies •Symmetry operations - most common to crystals of biological macromolecules are rotations, translations and screw axes Bravais Lattice Unit cell defined by cell edges A, B, C with angles α,β,γ angle between A and B is γ; B and C is α; A and C is β Triclinic Monoclinic A≠B≠C A≠B≠C α≠β≠γ not 90° α,γ= 90° β anything but 90° Orthorhombic A≠B≠C α, β, γ= 90° Hexagonal Rhombohedral Tetragonal A=A≠C A=B=C A=A≠C α, β = 90° α=β=γ≠90° α, β, γ= 90° γ=120° Cubic A=B=C α=β=γ=90° Symbols to Denote Symmetry Into the page Along the page ⟶ ⇀ 2-fold 2-fold screw axis 2-fold 2-fold screw axis 3-fold 3-fold screw axis 4-fold 4-fold screw axis Two-Fold Axes of Rotation Three-Fold Axes of Rotation Mirror Planes and Inversion Center Bravais Lattice ! •! Primitive (P): lattice points on the cell corners only.! ! ! •! Body (I): one additional lattice point at the center of the cell.! ! ! •! Face (F): one additional lattice point at the center of each of the faces of the cell.! ! ! •! Base (C): one additional lattice point at the center of each of one pair of the cell faces.! Space Group Projections - P222 Origin b c Origin Equivalent positions a a (x,y,z) (-x,-y, z) c Origin b (-x,y,-z) (x,-y,-z) a Origin b Projections - P2221 Origin b c Origin Equivalent positions a a (x,y,z) (-x,-y, z+1/2) c Origin b (-x,y,-z+1/2) (x,-y,-z) a Origin b Projections - P21212 Origin b c Origin Equivalent positions a a (x,y,z) (-x,-y, z) c Origin b (-x+1/2,y+1/2,-z) (x+1/2,-y+1/2,-z) a Origin b Projections - P212121 Origin b c Origin Equivalent positions a a (x,y,z) (-x+1/2,-y, z+1/2) c Origin b (-x,y+1/2,-z+1/2) (x+1/2,-y+1/2,-z) a Origin b Crystal Preparation for Data Collection • X-rays used for diffraction is ionizing. • Free radical formation of water molecules • Free radicals destroy crystal integrity, particularly at synchrotrons. • Flash-cooling crystals to liquid nitrogen temperatures reduces radiation damage. • Need to prevent the formation of crystalline ice • Add cryoprotectant to crystal Cryoprotectant • Selection of a suitable cryoprotectant involves some trial and error • A suitable cryoprotectant will cool to cryogenic temperature without ice formation, physical damage to the crystal, and preserve diffraction. • To assay for the proper concentration, mix the cryoprotectant with the crystallization reagent and add to crystal. • Examples - Glycerol, low MW PEG, Ethylene glycol, sugars, Butandiol, 2-Methyl-2,4- pentanediol Cryoprotection Loops “Home” Source - Rotating Anode National Synchrotron Light Source NSLS II – Coming Soon! NSLS NSLS II • $912 million to design and build • State-of-the-art facility that produces x-rays up to 10,000 times brighter than NSLS http://www.bnl.gov/ps/nsls2/about-NSLS-II.asp Experimental Floor at NSLS X29 Beamline NSLS Beam Stop Crystal Cold Stream X-rays Diffraction Pattern Radiation Damage Crystal after data collection Position of the beam is in red Diffraction Pattern.