99484 - Biomolecular Structure

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

Computational methods (Prof. 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.

Biocrystallography (Dr. Luca Mazzei):

Introduction (2 hours): Organization of lessons and learning assessment methods. Introduction to the program topics. Presentation of crystallography of biological macromolecules, its tools, and objectives.

Crystallization of biological macromolecules (4 hours): Thermodynamics and kinetics of crystallization of biological macromolecules, stages and mechanisms of the crystallization process and main techniques used for the crystallization of biological macromolecules.

Geometrical properties of crystals of biological macromolecules and physical bases of X-ray diffraction (6 hours): crystal symmetry and symmetry operations allowed for crystals of chiral molecules, definition of crystal unit cell, crystal lattices planes and lattice indexes, characteristics and production of X-rays, geometrical principles of diffraction and Bragg's law, techniques for X-ray diffraction data collection from crystals and properties of the collected data, such as positions and intensity of the reflections, data resolution.

Protein structure resolution methods using X-ray crystallography (6 hours). Structure factor as a Fourier sum, Fourier transform to obtain the crystal electron density, introduction to the phase problem and methods for its initial estimation: isomorphous replacement (SIR and MIR), anomalous dispersion (SAD and MAD), and molecular replacement (MR).

Obtainment of the final X-ray crystal structure and deposition (2 hours): improvement of the estimated phases, structural refinement and model building procedures, validation of the crystallographic structure and deposition on Protein Data Bank.

The course includes a practical part in laboratory (10 hours) consisting of three individual sessions, namely i) the crystallization of hen egg white lysozyme through the vapor diffusion method using different conditions, such as different precipitant types and concentration of protein, buffer and precipitant, ii) the examination of the obtained crystals under the optical microscope to assess the quality and choice of the best samples, and iii) the structural determination of the hen egg white lysozyme starting from experimental data collected from a lysozyme crystal and using the molecular replacement procedure.

Biomolecular NMR spectroscopy (Prof. Stefano Ciurli):

The course includes lectures and laboratory exercises with the aim of putting into practice the theoretical foundations of the technique. The syllabus includes an introductory lesson (2 hours) to present biomolecular NMR spectroscopy, its instruments and its objectives. The physical bases of NMR spectroscopy and the operating principles of the NMR spectrometer will be subsequently illustrated (6 hours). The following lessons (30 hours) will take place using single PC stations to better understand i) the NMR signal processing procedures for obtaining the NMR spectrum, ii) the chemical-structural information derived from the NMR spectrum of proteins , iii) the procedures for assigning signals and iv) the procedures for calculating the structure and dynamics of proteins in solution using NMR spectroscopy.

Readings/Bibliography

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

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

Bernhard Rupp “Biomolecular crystallography” Garland Science 2010

Ken Keeler “Understanding NMR Spectroscopy, Wiley, 2002

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

Andrew L. Leach “Molecular modelling” Pearson Education 2001

Teaching methods

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

Assessment methods

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.

Biocrystallography: the final exam consists of a test made of three questions and numerical problems, as well as a colloquium. At least one question of the test will cover the laboratory part. 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.

NMR spectroscopy: The final examination comprises a written test made of questions and numeric problems for students with a number of hours exceeding 90 % of the total. For all other students, the exam will consist of a colloquium.

 

Teaching tools

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 Luca Mazzei

See the website of Francesco Musiani