2. Protein Crystallography At SSRL By: Silpa Nalam and Andrea Kurtz Advisors: Aina Cohen, Paul Ellis, and Nick Sauter

3. Part 1: Introduction Project Description Protein Crystallization Web Page Design

4. Background on X-rays Advantages of SSRL x-ray beam lines over home x-ray tubes higher intensity tuning the x-rays allows one to choose one wavelength better resolution How is a diffraction pattern made? The pattern is made from diffracted x-rays by the atoms of the sample math and computers are used for interpretation because x- rays cannot be focused by a lens system dimensions of spots give unit cell information and their intensity gives the arrangement of the atoms in the unit cell Diffraction Pattern

5. What is a Protein? Basic Structure polypeptide chain and its average size is 10,000 Daltons R groups have 20 different combinations and help the protein bind to ligands, catalyze chemical reactions and direct the polypeptide in its conformation Functions enzymes, structural elements, antibodies, hormones, and electron carriers

6. Protein Structure 4 Levels of structure give the protein a 3-D shape which helps carry out its functions primary=sequence of R groups secondary=random coil, alpha, or beta structures tertiary=folding of secondary structures (biologically active) quaternary=more than one polypeptide chain Protein sensitivity denaturation (protein unfolding) caused by heat, chemicals, changes in pH Alpha Helix Beta Sheet

7. Common Proteins in the Body Myoglobin is used as an oxygen storage molecule that can be found in the muscle tissue of vertebrates Hemoglobin is a unique blood protein that carries oxygen from the lungs to the rest of the body. Leghemoglobin is a type of hemoglobin found in plants. It is an important part of the nitrate fixation process

8. Crystallization with X-rays Usefulness of x-rays wavelength of x-rays is 10 -8 cm Difficulties in Crystallization not usually in a crystalline fashion not suited to be in crystalline form protein has an extreme sensitivity Why Crystallization? provides the answer to many structure related questions

9. Background on Crystals A crystal is a periodic arrangement of unit cells in a lattice. The unit cell is repeated in a 3-D lattice to make a crystal. Common example of a crystal in a lattice is sodium chloride

10. Materials for Experiment Linbro Plate 24 well tray and each well is 1.5 cm in width and 1.8 cm in depth Linbro Plate 24 well tray and each well is 1.5 cm in width and 1.8 cm in depth Cryo Solutions Allows for the crystals to grow. They can be bought or homemade. Cryo Solutions Allows for the crystals to grow. They can be bought or homemade. Myglobin The protein is placed in a reagent that reduces its solubility to close to precipitation. The concentration should be 5-50 mg/mL Myglobin The protein is placed in a reagent that reduces its solubility to close to precipitation. The concentration should be 5-50 mg/mL Gel The gel is used to ensure the coverslips stay over the well Gel The gel is used to ensure the coverslips stay over the well

11. Procedure For Experiment Vapor-Diffusion Method The protein/precipitant mixture in the drop is less concentrated than in the well solution. Water evaporates from the drop until the concentration of the precipitant in the drop is the same as in the well. The conditions in the drop determine whether the protein will crystallize or not. Such as: ionic strength, temperature, and protein concentration. Finished Tray

12. Variables As the precipitant point of the protein is approached, factors such as pH, temperature, ionic strength, and choice of buffer control whether the protein will separate from the solution as a crystal or a precipitate. The pH, type of cryoprotectant (PEG or MPD) and buffer should be varied to get the protein in crystalline fashion. It is hard to crystallize proteins under normal conditions because the crystal might react with water vapor. The crystals are usually grown in a cold room.

13. Key Points Reproducibility Crystallography: art or science? Software based trials Crystallization process growing crystals analysis of crystals production of data conclusion theory of crystallization

14. Protein Crystallography at SSRL Part II: Determining Crystal Quality and Data Collection and Analysis

15. Background on Crystals A body that is formed by the solidification of a chemical element, a compound, or a mixture and has a regularly repeating internal arrangement of its atoms and often external plane faces 7 Classes of Unit Cells Described by three axes (a,b,c) and three angles ( , ,  ) Below: Packing diagram for orthorhombic unit cell of BPTI in the most common space group, P212121 Left: Single molecule of bovine pancreatic trypsin inhibitor (BPTI), considered the asymmetric unit in the unit cell below 230 Space Groups Characterized by symmetry elements such as rotational and screw axes The presence of symmetry elements simplifies data collection and analysis.

16. Macromolecular Crystallization Crystallization of the protein is only the first step…

17. Examination of Samples Protein remains solubleAmorphous precipitateGel precipitate Crystalline precipitateCyclophilin crystals

18. Crystal Quality at a Glance Characteristics of Crystals Suitable for Analysis: Large size, i.e., average length of approximately 200  m Single crystals with smooth, distinct faces and edges Color (in many cases) or opacity (for myoglobin) Common Protein Crystal Defects: Bunching or twinning Size deficiencies Denaturation with age

19. Mounting and Flash-Cooling Many protein crystals are extremely sensitive to changes in temperature and surrounding conditions (i.e., presence of original solvent). The crystal must be flash-cooled to 100K in a stream of liquid nitrogen to prevent damage from high-intensity x-rays. Crystals grown in the cold room must be flash-cooled and transported to the experimental hutch with cryo-tools. Teasing the crystal from the coverslip droplet Copper pin on goniometer head in liquid nitrogen stream

20. Data Collection Variables X-ray beam wavelength (on tuneable beam lines) Crystal-to-detector distance  = degree of oscillation around phi axis Exposure time Start and end  -angle According to the size of the crystal and the type of protein, the following factors are assigned prior to data collection: Sample Control Panel at Beam Line 9-1

21. The Diffraction Pattern Key Features: spot separation unit cell dimensions spot intensity unit cell contents Myoglobin diffraction pattern at 1.598Å resolution Left: Bragg’s Law and reflection of x-ray beams from crystal planes

22. Origins of the Diffraction Pattern

23. Data Interpretation Using Mosflm, we can perform the following functions on the data embedded in the diffraction pattern: Autoindexing and Cell Refinement unit cell determination mosaicity orientation matrix Integration intensity: count-integrated and profile-fitted on each image combine reflections on multiple images Scaling and Merging combining equivalent reflections crystal breakdown and beam decay The importance of resolution in structural determination The importance of resolution in structural determination Tests of Data Quality: R-factor Percentage of data measured Systematic absences I/  I and F/  F

24. Methods of Solving Structures Direct - small molecules (less than 100 amino acids) with very high resolution data Multiple Isomorphous Replacement (MIR) Multiwavelength Anomalous Dispersion (MAD) Molecular Replacement Method (MRM) Above: Fourier synthesis diagrams. The red lines represent the model that was used to find the actual amplitudes and phases. Both factors are essential, since phases are not directly measurable and must be calculated from amplitudes Random Amplitudes Random Phases

25. Future Improvements Automated Batch Handling and X-Ray Diffraction Analysis Integration of Computer Technology with Human Ability Increased Internet-Based Interfacing for Data Collection, Analysis, Storage, and Retrieval Crystallographer’s Creed This is my x-ray machine. There are many like it, but this one is mine. My x-ray machine is my best friend. It is my life. I must master it as I must master my life. Without me, my x-ray machine is useless. Without my x-ray machine, I am useless. I must collect my data true. I must phase faster than my enemy who is trying to publish before me. I must shake him before he bakes me. I will. -Dr. Bernhard Rupp, LLNL