Synchrotron Science and Protein Crystallography

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1 Your Brief You will work with your mentors to grow protein crystals and use X-ray diffraction sources to study the crystal structure of lysozyme. You will have an overall view of what it takes to study protein crystallography. Your Report You will produce a report about your project. This can be done as a group with each person contributing or you can do individual reports. This will take the form of a poster (the template is provided) and a newsletter article (about words) for your school and the lab to use in their newsletters. Please include group photos with full names of members. The report should include some of the basic science and an outline of your learnings about: Why this research is important How it is connected to real world problems and how can it help society, individuals Why high school students should care about the physical sciences/potential career paths How it has or can help you keep engaged with your study of science and especially physics and mathematics. Your Presentation You will also work together to present a powerpoint presentation. It only needs to be short, and the template has been provided. You will include: How working with the scientists has helped changed the way you learnt the science, and how it felt if it worked or not Scientific questions you had/posed for investigation during the week - even ones that did not get answered How your perception of the physical sciences, scientists and even career pathways may have changed

2 Introduction The Australian Synchrotron is an electron accelerator the size of the MCG. It uses electricity to produce intense beams of light a million times brighter than the sun. Scientists use this high energy radiation in a number of ways at various experimental workstations, with applications ranging from medical imaging to looking at artwork to investigating the structure of materials. As part of this project you will learn how the synchrotron and X-rays can be used to see inside molecules and work out their atomic structure using a technique called X-ray crystallography. This is the same technique that was used to find the double-helix structure of DNA. High energy electron sources are used in the sciences, especially the biological sciences to observe the unseeable. Knowing the structure, i.e. the shape and size of objects, means that we can understand them and compare them to other things. It allows us to organise information and find patterns. This can be useful for understanding how biological process work and why they stop working and how to fix or inhibit them. You will be setting up and carrying out a real protein crystal-growing experiment in a laboratory using lysozyme. Lysozyme is an enzyme found in humans, animals and plants. This enzyme is present in mucus, tears, saliva, sweat and human milk and it is responsible for breaking down complex sugars by breaking the connections between individual simple sugar molecules that hold them together. Lysozyme has an antibacterial role by breaking down sugars found in the bacterial cell wall, resulting in the death of the bacteria and thus protecting our body from infections. In this project we will be working with lysozyme from the chicken egg white. In 1909, Laschtschenko first observed the antibacterial nature of a protein in egg white. However, it was only in 1922 that Alexander Fleming named this protein Lysozyme due to its ability to lyse the cells of many microbial species. Fleming demonstrated the action of lysozyme by treating bacterial cultures with mucus taken from a patient with a head cold and observing the inhibition or destruction of bacteria around it. In 1965, Lysozyme was the first enzyme to have its 3D structure solved by X-ray Crystallography. David Chilton Phillips was responsible for this work and it took him many years between obtaining crystals, to reaching the final structure. This study provided the first explanation for how enzymes physically catalyse (speed up) chemical reactions. We will shoot the lysozyme crystals that we obtain with X-ray beams and the diffraction data will be collected to solve the structure of this enzyme. Luckily, the process of data collection and structure determination has rapidly advanced since then and now it can take a minimum of 1 month to carry out this process. However, we will use computer programs to solve the structure of the protein in 1 day. The importance of this technique is that it can help guide the development of therapeutic compounds, like antibiotics and anti-cancer drugs, as well as help with understanding how biological processes work, such as iron uptake. Can you think of other ways this is important? Come with questions to contribute to your project development. :) X-ray crystallography has been used for centuries to solve structures of proteins which are ~2 nm big. Other things can also be used like Cryo EM and NMR. There are other electron sources at

3 the Australian synchrotron including SAXS, WAXS, IR which are also used to structurally study proteins and molecules. Checkout the site below to find out what this is and how it works. Lysozyme Have a look at this link if you would like to know more about Lysozyme If you would like a detailed description of how Lysozyme functions watch this video Schedule Monday Meet at La Trobe University Library, 9.00am 9:00-9:30 Safety Induction TLC Dry Lab (TLC 106) 9:30-10:30 Brainstorm Session TLC Dry Lab (TLC 106) 10:30-12:00 Introduction and Growing Lysozyme Crystals TLC Dry Lab (TLC 106) 12:00-1:00 Lunch break 1:00-2:00 The Australian Synchrotron and MX Beamlines LIMS :00-4:00 Presentation Preparation Tuesday Meet at La Trobe University Library, 9.00am 9:00-9:05 Welcome Back Library 9:10-10:30 X-ray Data Collection LIMS X-ray lab 10:30-12:00 Data Analysis LIMS :00-1:00 lunch

4 1:00-2:00 Campus Tour Library 2:00-4:00 Presentation Preparation - Posters to be completed by end of session Wednesday Meet at La Trobe University Library, 9.00am 9:00-9:05 Welcome Back Library 9:15-10:45 AgriBio Tour AgriBio 11:00-12:00 Presentation Preparation 12:00-1:00 Brainstorm Session 1:00-1:30 Poster session 1:30-2:00 Lunch 2:00-4:00 Presentations PS1 114 (VisLab)

5 ` Protein Crystallography Synchrotron Science and Protein Crystallography Crystal growing workshop Learning objectives Prepare a table with formulations for a crystallisation screen. Prepare a table with dilutions to prepare samples of varying protein concentrations. Set up standard crystallisation experiment. View the result of a crystallisation experiment with a stereomicroscope. Understand the principle of protein crystallography. The basis of a crystallization experiment In this practical you will provided with a standard crystallisable protein (Lysozyme). You will need to design a crystallisation experiment, where the concentration of precipitant agent (PEG 6000 and NaCl) and protein (Lysozyme) are varied in a systematic manner across 4 of the 24 wells of the crystallisation plate. You will set down 4 sitting drops as 1:1 mixtures of protein and reservoir solution. At the end of the session you will observe the results of these experiments under the stereomicroscope. Materials and Reagents Lysozyme stock solution at 100 mg/ml 1.5 ml of crystallisation cocktail (30% w/v polyethylene glycol 6000, 1M Sodium chloride, 0.05M Sodium acetate trihydrate ph 4.6) 1 ml of distilled water 24-well VDX plate Sealing tape Setting up lysozyme crystals 1. Put on a lab coat, safety glasses and gloves 2. Label one column (A-D) of the 24-well plate with your name 3. Prepare strips of sealing tape that will cover each of the wells 4. Prepare the reservoir solutions by diluting a series of crystallisation cocktail (carefully pipette up and down to mix the diluted cocktail before proceeding to the next well)

6 Well A 200µL Crystallisation cocktail Well B 200µL water + 900µL Crystallisation cocktail Well C 200µL water + 900µL from well B reservoir Well D 200µL water + 900µL from well C reservoir 5. Add 2µL of lysozyme in the centre of the raised platform in each well (carefully pipette the reservoir solution directly on top of the protein drop without introducing air bubbles).

7 6. Seal the well with the tape strip 7. Incubate at room temperature for 10 minutes 8. View the plates under the stereomicroscope