D1, KNOWLEDGE AND UNDERSTANDING: Know the basic elements of protein structures. Acquire an overview of the main sample preparation techniques for structural biology analysis. Learn the features of crystals and the main crystallization techniques. Understand the physical basis of X-ray diffraction and acquire knowledge of data collection techniques, structural determination, and structure refinement used in biocrystallography. Understand the relation between the real space and the reciprocal space. Acquire a basic knowledge of electron microscopy techniques applied to structural biology. Learn the basic principles of analysis of electron microscopy data, leading to the determination of the 3D structure of a protein. Acquire the basic principles and tools to understand the relations between the structure of proteins and their biological function. D2, APPLYING KNOWLEDGE AND UNDERSTANDING: Describe the 3D structure of proteins through a systematic and hierarchical description of the elements characterizing protein folding. Design a biocrystallographic experiment, including expression, purification, crystallization, and diffraction data collection. Analyze diffraction data from protein crystals, from diffraction images to the complete and refined 3D protein structure. Identify the steps required to obtain a 3D structure from electron microscopy data. Evaluate a structure obtained from biocrystallography or electron microscopy in terms of the correctness of the global and local protein fold and reliability. D3, MAKING JUDGMENTS: Identify the motives behind the selection of different expression, purification, and crystallization methods, and evaluate how to apply them to specific protein specimens. Recognize crucial factors that can improve/hamper a structural biology experiment. Identify the advantages and disadvantages of biocrystallography or electron microscopy techniques. Identify significant information that can be obtained from a protein structure and the experimental factors that can influence the results. Evaluate the quality of a protein structure obtained through crystallographic or electron microscopy techniques. D4, COMMUNICATION SKILLS: Present a structural biology paper, using the specific terminology acquired during lectures. Highlight the useful information that can be extracted from each result. Identify and explain the structure-function relationships recognized thanks to the structural biology techniques. Produce images that help visualize the interesting aspects of a protein structure, using the software described during lectures and applied in laboratory experiences. D5, LEARNING SKILLS: Read and understand a paper on structural biology subjects and discuss its critical points. Know and consult the principal IT resources mentioned during lectures, relative to the structural biology field.

General Chemistry. Biochemistry. Mathematics. Physics.

Introduction to structural biology: structure-function relationship. Principles of protein structure. Data banks of proteins. Software for the visualization of protein structures. Expression of recombinant proteins for structural studies. Basic molecular biology techniques. Methods for the purification of proteins. Methods for the evaluation of sample quality, purity, conformational stability, and oligomeric state. Protein crystals. Thermodynamic and kinetic aspects of crystallization. Crystallization techniques. Symmetry in crystals. Symmetry elements and periodicity of the crystal lattice. Space groups. Non-crystallographic symmetry. Miller indexes. Reciprocal lattice. X-rays. Scattering from atoms. Argand diagram and interference. Scattering from the crystal lattice (Bragg's law). Ewald sphere. Symmetry in the reciprocal space. Absorption of X-ray and anomalous scattering. Instrumentation. Strategies for data collection. Radiation damage. Evaluation of data quality. The phase problem. Methods for solving the phase problem for biological macromolecules: molecular replacement, density modification, isomorphous substitution, and anomalous dispersion methods. Refinement of the structural model. Evaluation of model quality and validation. Transmission electron microscopy in structural biology. The Electron Microscope. Image formation in the weak phase object approximation. Contrast Transfer Function (CTF). Electron crystallography. Reciprocal space for 2D crystals. Diffraction data analysis and image analysis for 2D crystals. Single-particle techniques in electron microscopy. Image classification, alignment, and averaging. 3D model reconstruction from electron microscopy images. Statistical methods with the use of Bayesian probability. Validation. Practical experiences in crystallography (protein crystallization, structure solution and refinement) and electron microscopy (negative staining sample preparation, data analysis). Model analysis using molecular graphics software.

Bernhard Rupp, "Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology", editor Garland Science.
Joachim Frank, "Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State", editor Oxford University Press.
Carl Branden and John Tooze, "Introduction to Protein Structure", editor Garland Science.
Lectures' slides are available on Moodle, together with short movies that help visualize the protein structures mentioned during theoretical lectures. Also available on Moodle: links to free versions of software and databanks used during practical experiences. Further bibliographical sources (papers and reviews) are suggested at the end of lectures.

Introduction to structural biology: structure-function relationship. Principles of protein structure. Primary structure, secondary structure, tertiary structure, and quaternary structure, with examples. Post-translational modifications and cofactors. Membrane proteins. Active sites and domains. Data banks of proteins. Software for the visualization of protein structures. Expression and purification of recombinant proteins for structural studies. Bioinformatic resources. Choice of expression system. Construct design. Basic molecular biology techniques. Purification. Methods for the evaluation of sample quality and purity. Analysis of conformational stability. Analysis of protein oligomeric state. Protein crystals. Thermodynamic and kinetic aspects of crystallization. Crystallization techniques for soluble and membrane proteins, optimization strategies. Co-crystallization and soaking. Automation in the crystallization experiment. Symmetry in crystals. Rotation and screw axes. Periodicity of the crystal lattice. Unit cells and crystal systems. Bravais lattices. Space groups. Fractional coordinates. International Tables for Crystallography. Asymmetric unit. Non-crystallographic symmetry. Miller indices. Reciprocal lattice. X-rays. Scattering from electrons and atoms. Argand diagram and interference. Scattering from the crystal lattice and Bragg's law. Ewald sphere. Friedel pairs. Symmetry in reciprocal space. Laue groups. Systematic absences. Absorption of X-ray and anomalous scattering. Instrumentation. Conventional sources. Synchrotron beamlines for crystallography. Optics. Goniometer. Detectors. Cryocrystallography. Strategies for data collection. Radiation damage. Twinning. Data analysis. Evaluation of data quality. The phase problem. Methods for the solution of the phase problem for biological macromolecules. Patterson function. Molecular replacement. Density modification methods. Isomorphous substitution method. Anomalous dispersion method. Electron density maps. Refinement of the structural model. Evaluation of model quality and validation. Transmission electron microscopy in structural biology. The Electron Microscope: source, electromagnetic lenses, goniometer, detector. Image formation. Weak phase object approximation. Contrast Transfer Function (CTF). Electron crystallography. 2D crystallization of membrane proteins. Sample preparation: negative staining and cryo. Data collection. Reciprocal space for 2D crystals. Diffraction data analysis and image analysis for 2D crystals. Single-particle techniques in electron microscopy. Sample preparation. Signal and noise. Sampling of the digital signal. Image classification, alignment, and averaging. Cross-correlation function. 3D model reconstruction from electron microscopy images. Orientation of the particles: Euler angles. Ab initio methods: random conical tilt and common lines. Methods of reconstruction by comparison with a model. Statistical methods with the use of Bayesian probability. Validation. Practical experiences: Crystallization through vapor diffusion techniques. Diffraction data analysis: data reduction, structure solution, and refinement. Negative staining sample preparation for electron microscopy. Cryo-EM data analysis. Model analysis using molecular graphics software. Image preparation.

Traditional frontal lectures with slides and interactive sessions using free software to visualize protein structures. Laboratory experiences in which each student familiarizes with the main crystallization techniques, sample preparation, and data analysis (data reduction, structure solution, and refinement). The lecturer promotes interaction with the students during lectures and practical sessions. Students' questions will be answered during the course. Presentations used during lectures will be available in advance on the Moodle platform of the University.

Links to free resources relative to subjects covered in the lectures are available on the Moodle platform, together with slides used during lectures. Students are encouraged to consult volumes of the International Tables of Crystallography available at the lecturer's office or in the library, to deepen their knowledge of space groups and symmetry elements of crystal structures. For clarification on the subjects of the course, students can take an appointment with the lecturer by email (rdezorzi@units.it) and meet at the lecturer's office (building C11, room 427).

Oral examination with a slide presentation and discussion of a scientific paper reporting the structure of a protein determined using X-ray crystallography or electron microscopy. In preparing the presentation, the student will consult available data banks and obtain the structure(s) of the protein(s) studied in the paper. The student will be evaluated on his/her ability to explain results obtained by the authors, using figures to highlight the main findings. Specific questions by the examiners will verify the student's knowledge of the basic theoretical concepts explained during lectures (crystal symmetry, diffraction theory, electron microscopy data analysis, etc.). The ability of the student to design a structural biology experiment and identify its critical steps for a positive outcome will be assessed. The student will be asked to comment on possible issues in the research presented, identify the weaknesses, and devise a strategy to answer the remaining open questions. The result of the exam will be evaluated with a score of thirty points based on the following criteria: -Excellent (30-30 cum laude): excellent knowledge of the topics, excellent language skills, excellent analytical skills; the student is able to brilliantly apply theoretical knowledge to concrete cases. -Very good (27-29): good knowledge of the topics, remarkable language skills, good analytical skills; the student is able to correctly apply theoretical knowledge to concrete cases. -Good (24-26): good knowledge of the main topics, good language skills; the student shows adequate ability to apply theoretical knowledge to concrete cases. - Satisfactory (21-23): The student does not fully master the main topics of teaching, but has the basic knowledge; however, he/she demonstrates satisfactory language skills and an adequate ability to apply theoretical knowledge to concrete cases. -Sufficient (18-20): minimal knowledge of the main topics of teaching and technical language, limited ability to adequately apply theoretical knowledge to concrete cases. - Insufficient (less than 18): The student does not have acceptable knowledge of the content of the various topics of the teaching course. Any changes to the methods described here that may be necessary to ensure the application of safety protocols in the context of possible emergency situations will be announced on the Department website, the Course of Study and on the Moodle page for teaching.

Lectures will not discuss subjects related to the Objectives of the 2030 agenda for sustainable development.