Crystallography basics - Crystallization Josef Houser Autumn 2023 S1004 Methods for structural characterization of biomolecules Crystal Crystal lattice Bravais lattice • P – primitive • I – body centered • C – base centered • F – face centered • 14 together Translational symmetry • Essential property of crystal Unit cell Symmetry • Ability of object to stay the same after transformation • Symmetry operations: • Translation – vector • Reflection – plane • Rotation – axis TRANSLATION REFLECTION ROTATION TRANSLATION ROTATION Reflection symmetry • One half of object is mirror image of the other • Plane of symmetry ≠ n = 1 n = 3 n = 4 n = 1n = ∞ Reflection symmetry • Object may have several reflection symmetries etc. Rotational symmetry • Degree of symmetry is the number of distinct orientations in which it looks exactly the same • Axes of symmetry n = 1 n = 3 n = 4 n = 1n = ∞ identity identity Rotational symmetry • Object may have several axes of symmetry A B CD AB CD AB C D AB CD C2 C4 (C1) Rotational symmetry • How many symmetry axes (and which) has cube? 3x 4-fold 4x 3-fold 6x 2-fold Combined symmetries • Rotation + translation = screw-axes • Reflection + translation = glide plane • Reflection + rotation = rotoinversion Point groups • Set of symmetry operations that can be applied to the crystal with the fixed initial point • 32 existing point groups for crystals Space groups Example of space group information from International Crystallographic Tables Combination of Bravais lattices and symmetry operations leads to 230 possible space groups. Asymmetric unit • Smallest part of crystal that can be used to reconstruct crystal based on crystal symmetry and translation pdb.rcsb.org Biomacromolecular crystal • Not all symmetry operations possible Amino acids Carbohydrates (and Nucleic acids) are CHIRAL Reflection symmetry is not applicable – no mirrors Only 65 space groups exist for protein Biomacromolecular crystal XDS Package Biomacromolecular crystal • Properties for crystals of small molecules and biomacromolecules differ Small molecule crystal Biomacromolecular crystal Size < 1 dm < 1 mm Composition Localized molecules/ions (Coordinated water molecules) Localized macromolecules Free ions/water molecules Stiffness Moderate – Hard Soft Temperature stability Frequently up to melting Frequently only within few degrees Growth Usually quick and spontaneous Usually slow or problematic Biomacromolecular crystal • Biomolecular crystals contain 25 – 80 % of solvent • Matthews coefficient VM = V(cryst) / 1 Da(prot) • “Oriented gel”-like 1 nm Solvent channels Protein Crystallization • Process of self-arranging of molecules into crystal lattice • Thermodynamics – enthalpy, entropy, kinetics Saridakis 2008 ~ r2 ~ r3 Wang 2016 Phase diagram • Existence of various phases in the system depending on variables • Phase: gas, liquid, solid • Variable: temperature, pressure, composition Phase 1 Phase 2 Variable 1 Phase 3 Variable 2 Phase diagram https://www.tf.uni-kiel.de/ https://www.chegg.com/ http://ergodic.ugr.es/ Protein phase diagram – T vs. conc. • Solubility increase with temperature • Limited application due to protein conformational instability at higher T http://ppl.ippt.gov.pl/solution-crystallization Fabrice Gorrec Protein phase diagram – composition • Precipitant: • Buffer • Salt • Small organics • Mixtures Real phase diagram • Lysozyme vs. NaCl in acetate buffer c(lysozyme) c(NaCl) c(lysozyme) c(NaCl) www.xray.bioc.cam.ac.uk Crystallization techniques • Small molecules • In situ crystallization • Cooling • Solvent evaporation (free, under vacuum) • Solvent exchange jinachem.blogspot.comchemhelper.com chemistryviews.comchemistryviews.com Crystallization techniques • Small molecules • In situ crystallization • Cooling • Solvent evaporation (free, under vacuum) • Solvent exchange • Macromolecules • Batch • Vapor diffusion • Dialysis • Free interface diffusion Fabrice Gorrec Batch method • Direct mixing of protein and precipitant • Drop under paraffine oil – prevention of evaporation Fabrice Gorrec Vapor diffusion • Increasing of protein and precipitant concentrations due to H2O evaporation • Sitting drop vs Hanging drop vs Sandwich Fabrice Gorrec Precipitant: c1 > c2 c1 c2 c2 Under oil vapor diffusion • Modification of vapor diffusion • Slowing down the process • Paraffin oil vs. Silicon oil • Speed adjustment by paraffin oil : silicon oil ratio Fabrice Gorrec Dialysis • Increase of precipitant concentration due dialysis through semipermeable membrane • Constant protein concentration Fabrice Gorrec Precipitant: c1 > c2 (=0) c1 c2 semipermeable membrane Diffusion • Diffusion of protein and precipitant in opposite direction • Free-interface in capillary or Unidirectional diffusion – similar to dialysis Fabrice Gorrec ? porous material (gel) precipitant protein McPherson 2013 Seeding Stimulation of crystallization by introducing crystallization nucleus • Macro seeding • Micro seeding • Streak seeding • Cross seeding Saridakis 2008 http://xray.bmc.uu.se “bad” crystal crushing dilution seeding “good” crystal Micro seeding Seeding specials • Universal nucleant • Imprinted polymers N.Chayen Derivatization • Modification of crystallized protein • Ligands/substrates • Co-factors • Inhibitors • Heavy-atom compounds • Techniques • Co-crystallization • Soaking Co-crystallization • Adding of ligand BEFORE crystallization • Ligand in excess – homogenous complex needed • Binding not affected by crystallization • Crystallization affected (or blocked) by binding • Change of space group • Automation compatible Soaking • Adding of ligand AFTER crystallization • Ligand in various concentrations • Crystallization not affected by binding • Binding affected (or blocked) by crystal formation • Usually manual • High-throughput possible Heavy atoms derivatization • Introduction of heavy atom for phasing Compounds routinely available Compounds available on special demand Heavy atom derivatization at BIC Membrane proteins crystallization • Detergents • Liquid cubic phase (LCP) • Sponge phase Crystallization plastics • Standardized footprint formats (SBS, VDX) • 24/96 wells • high-throughput/automation • Various layouts • UV transparent Commercial screens • 48 or 96 conditions screens (deep well or tubes) • Various purposes – sparse screens, systematic screens, optimization screens, additive screens, ... • Producers: • Molecular Dimensions • Hampton • Jena Biosciences • Qiagen • ... Robotics for crystallization • Precipitant mixtures preparation • Drops set-up • Storage and imaging • (Crystal harvesting) Automatization vs. manual work ❑ High-throughput ❑ Low volumes (20-150 nl) ❑ Reproducibility ❑ Individual design ❑ Immediate visual control ❑ Complex sample handling Further reading • http://journals.iucr.org/ • Naomi E. Chayen: Protein Crystallization Strategies for Structural Genomics, 2007 • Terese M. Bergfors: Protein Crystallization, 2009 • Alexander McPherson: Introduction to Macromolecular Crystallography, 2011 • etc. Questions? 45 ? CF Head Josef Houser • +420 549 492 527 • josef.houser@ceitec.cz bic@ceitec.cz bic.ceitec.cz Biomolecular I nteractions and Crystallography Core Facility