Course Unit Page

Academic Year 2021/2022

Learning outcomes

At the end of the course, the student will have the basic knowledge necessary for the determination of the structure of biological macromolecules at the atomic/molecular level by using the three main techniques - X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR), and computational methods - and will be able to critically evaluate the opportunity to use such techniques. In particular, the student will be able to: i) interpret the results of an X-ray structural investigation, ii) develop the skills necessary for a critical reading of biocrystallographic papers, and iii) apply the main crystallization techniques of proteins through practical activities. Furthermore, the student will acquire familiarity with the theoretical and the experimental aspect of NMR spectroscopy, and in particular knows: i) the physical and theoretical basis of NMR spectroscopy, ii) the NMR experiments used to for the determination of the structure and the dynamics of proteins, and iii) the approach based on NMR spectroscopy for the study of molecular interactions. At the end of the course, the student will grasp the basics of the theory and practice of NMR spectroscopy, and their applications for the determination of protein structures. Finally, the student also acquired theoretical and practical knowledge on the calculation and the dynamics of macromolecules e macromolecular complexes through techniques of computational chemistry applied to proteins and metallo-proteins.

Course contents

Biocrystallography (Dr. Simona Fermani):

Introduction (2 hours): Organization of lessons and learning assessment methods. Introduction to the program topics. Introduction to biocrystallography, with particular reference to its tools, objectives and potentials.

Crystallization of biological macromolecules (4 hours): Basic principles, stages and mechanisms of the crystallization process with particular focus on properties and growth of protein crystals. Main techniques for the crystallization of biological macromolecules.

Physical bases of X-ray diffraction and elementary cells of a crystal (6 hours): Characteristics of X-rays, conventional sources and synchrotron light, techniques for collecting the intensity of diffraction from protein crystals. Geometrical principles of diffraction and Bragg's law. Symmetry in the crystals and symmetry operations allowed for chiral molecules. The unit cell and the indexes of the reticular planes in a crystal, positions and intensity of the reflections, data resolution.

Crystallography phase problem and protein structural resolution methods (8 hours). Structure factor as a Fourier sum; Fourier transform to obtain the electron density of the molecule under examination. Introduction to the phase problem and methods for the determination of the phase: molecular replacement, isomorphous replacement (SIR and MIR), anomalous scattering (SAD and MAD, SIRAS and MIRAS). Improvement of the phases and refinement of the model. Validation of the crystallographic structure and deposition on Protein Data Bank.

The course includes a practical part in laboratory: Crystallization of a model protein by elaborating an individual work procedure based on some data such as precipitant type and concentration of protein, buffer and precipitant. Examination of the crystals obtained under the optical microscope to assess the quality and choice of samples suitable for RX investigation. Exercise with use of Coot software suitable for visualizing the obtained protein model, electron density maps, map fitting processes and model regularization.

Biomolecular NMR (Prof. Stefano Ciurli):

Nuclear magnetism, interaction between dipoles and magnetic fields, quantization of the magnetic moment, Larmor frequency, precession of nuclear spins, nuclear relaxation. CW-NMR and FT-NMR spectroscopy. The parameters of the NMR spectrum: chemical shift of typical amino acids, chemical shift index, signal intensity and relaxation, scalar coupling and spin multiplicity, dipolar coupling and NOE effect. NMR spectroscopy in two dimensions: COSY, TOCSY and NOESY of amino acid residues in proteins. Multidimensional NMR spectroscopy: NOESY-TOCSY, HSQC, NOESY-HSQC. Sequence-specific assignment. Triple resonance spectra. Calculation of protein structure: principles and methodologies. Processing of NMR data: principles and methodologies.

Computational methods (Dr. Francesco Musiani):

Introduction (1 hour): Organization of lessons and learning assessment methods. Introduction to the program topics. Presentation of the computational structural biology, its tools and its objectives. Molecular mechanics and empirical force fields (3 hours): Recall to atomic models and to the concept of chemical bond. Coordinate systems. Molecular mechanics. Representation of atoms and molecules. Potential energy surfaces. Empirical force fields. Electrostatic interactions. Intermolecular interactions. Van der Waals forces. Polarizability. Hydrogen bonds. Energy minimization. Molecular dynamics (theory, 2 hours): Methods for the exploration of the potential energy surface. Simulation methods. Molecular dynamics and outline of methods for the accelerated exploration of the potential energy surface. Simulation box. Implicit and explicit solvent. Statistical ensembles. Thermostat and barostat. Homology modelling and docking (theory, 2 hours): Principles of homology modeling. Data base of protein sequences. Sequence alignment. Prediction of the protein structure. Loop modeling. Molecular recognition (docking) between a protein and a small molecule and between a protein and another biomolecule. Evaluation of the models. Homology modelling exercise (4 hours): Introduction to the Linux operating system. Review of the use of the UCSF Chimera molecular visualization software. Application of the software Modeller for the modeling of two metallo-protein. Optimization, evaluation and analysis of the obtained models. Molecular recognition exercise (4 hours): Use of the DOCK program docking of a small molecule. Use of the Haddock webserver and protein-protein protein-protein docking. Evaluation and analysis of results. Molecular dynamics exercise (4 hours): Use of the GROMACS program. Setup of a molecular dynamics system in explicit solvent. Minimization, solvent equilibration and production dynamics. Analysis of the results.


Gale Rhodes "Crystallography made crystal clear" III ed., Academic Press 2006

Alexander McPherson "Introduction to macromolecular crystallography" J. Wiley 2003

Ken Keeler “Understanding NMR Spectroscopy, Wiley, 2002

Andrew L. Leach “Molecular modelling” Pearson Education 2001

Bertini,McGreevy,Parigi "NMR of Biomolecules", Wiley.

Teaching methods

Classroom lectures will be given by using slide presentations, in addition to laboratory sessions.

Assessment methods

Biocrystallography: the final exam consists of an oral colloquium to assess the students' knowledge of the topic. The oral exam will be preceded by a presentation, in which the student will have to comment on the results obtained from the laboratory experiences.

NMR: The final examination comprises a test made of questions and numeric problems, as well as a colloquium. Access to the colloquium is not prevented if the test is failed, but the test outcome will be considered to determine the final grade. No partial tests will be carried out through the teaching period.

Computational methods: the final exam consists of an oral interview to assess students’ knowledge of the topic. The oral exam will be preceded by a presentation, in which the student will have to discuss a scientific article concerning the module program and assigned in advance. Moreover, the student will have to answer questions concerning the presentation and the program of the module.

Teaching tools

Laboratory experiment of protein crystal growth, choice of the best sample, X-ray data collection and model building.

The multimedial material utilized during the lectures will be made available for download from the teacher's web site.

Office hours

See the website of Stefano Luciano Ciurli

See the website of Simona Fermani

See the website of Francesco Musiani