C8507 - Structural Determination and Computational Modelling of Biological Macromolecules

Learning outcomes

Upon completion of the course, students will have acquired basic knowledge for determining the three-dimensional structure of biological macromolecules using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and computational modeling. Specifically, students will develop an understanding of the theoretical and experimental principles of X-ray crystallography and cryo-EM applied to biomolecules (proteins and nucleic acids) through a combination of lectures and practical laboratory activities. Furthermore, students will acquire theoretical and practical skills in using computational chemistry methods to study the structure and dynamics of proteins and metalloproteins. Finally, students will be able to critically evaluate the suitability of these methods for structural analysis and will develop the critical skills necessary to analyze the scientific literature related to the study of models of biological macromolecules obtained through crystallographic, cryo-EM, and computational techniques.

Course contents

Module 1. X-ray crystallography and cryo-EM (Teacher: Dr. Luca Mazzei)

Introduction (2 hours). Organization of the module and learning assessment methods. Introduction to X-ray crystallography and cryo-electron microscopy (Cryo-EM) in the context of the structural determination of biological macromolecules.

Crystals of biological macromolecules (6 hours). Thermodynamics and kinetics of crystallization of biological macromolecules. Stages and mechanisms of the crystallization process. Main techniques used for the crystallization of biological macromolecules. Crystal symmetries of chiral molecules. Definition of the crystal unit cell. Crystal lattice planes and Miller indices (lattice indexes).

Physical bases of X-ray diffraction (3 hours). Characteristics and production of X-rays for structural biology. Geometrical principles of diffraction and Bragg's Law. Techniques for X-ray diffraction data collection from biological macromolecular crystals. Properties of collected diffraction data, including reflection positions, intensities, and resolution. Structure factor. Application of the Fourier transform to obtain the electron density map of the crystal.

Protein structure resolution methods using X-ray crystallography (3 hours). From the structure factor to the electron density: an introduction to the phase problem and the main methods for its initial estimation, including isomorphous replacement (SIR and MIR), anomalous dispersion techniques (SAD and MAD), and molecular replacement (MR).

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

Fundamentals of cryo-electron microscopy (2 hours). Fundamentals of cryo-electron microscopy (cryo-EM), and a historical context from X-ray crystallography to Cryo-EM for the structural determination of biological macromolecules. Major applications of cryo-EM.

From the biological macromolecule sample to the cryo-EM structure (3 hours). Sample preparation for cryo-EM. Components of an electron microscope. Data collection methods, image analysis processes, and three-dimensional reconstruction. Model building, validation and case studies.

Laboratory (13 hours). Crystallization of a model protein through the vapor diffusion method using different conditions, such as different precipitant types and concentration of protein, buffer and precipitant. Examination of the obtained crystals under the optical microscope to assess the quality and choice of the best samples.
Determination of the X-ray crystal structure of the model protein starting from experimental data and using the molecular replacement procedure.

Modeule 2. Computational chemistry of proteins and metallo-proteins (Teacher: Prof. Francesco Musiani)

Introduction (1 hour). Organization of the module and learning assessment methods. Introduction to computational chemistry, computational structural biology and computational structural modelling.

Molecular mechanics (4 hours). A review of the concepts of atomic models, chemical bonding, intermolecular interactions and protein structure. Molecular mechanics, representation of atoms and molecules, empirical force fields and their limitations, additivity and transferability principles. Potential energy surfaces, energy minimisation, exploration of the potential energy surfaces, classical molecular dynamics, thermodynamic ensembles, thermostats and barostats, periodic boundary conditions, computational cost and parameter tuning. Free energy estimation and outlines of enhanced sampling of the potential energy surface.

Homology modelling (2 hours). Protein folding and evolution, homology modelling of proteins, improving and evaluating models, and applying machine learning algorithms to protein modelling.

Exercitations (12 hours). A historical overview and basic usage of the Linux operating system and the computer command line. A practical overview of the main command line commands. Overview of GROMACS software. Setup of the molecular dynamics simulation of a protein using an atomistic force field and explicit solvent. Molecular dynamics simulation and analysis of the results. Homology modelling of a protein and of a protein complex using classical and machine learning techniques.

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

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

X-ray crystallography and cryo-EM: The final examination will consist of a colloquium to verify the student's proficiency

Computational chemistry of proteins and metallo-proteins: 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.

Students with learning disorders and/or temporary or permanent disabilities: please, contact the office responsible (https://site.unibo.it/studenti-con-disabilita-e-dsa/en/for-students) as soon as possible so that they can propose acceptable adjustments. The request for adaptation must be submitted in advance (15 days before the exam date) to the lecturer, who will assess the appropriateness of the adjustments, taking into account the teaching objectives.

Teaching tools

The multimedial material utilized during the lectures will be made available for download from the e-learning portal Virtuale.

Students are asked to report any needs to the teacher via private mail. This will allow the teacher to evaluate which teaching support tools are most adequate to make the course accessible to all students.

Office hours

See the website of Francesco Musiani

See the website of Luca Mazzei