Structural Biology

Teachers: 
LUISI BONAVENTURA FRANCESCO
Credits: 
6
Site: 
PARMA
Year of erogation: 
2021/2022
Unit Coordinator: 
Disciplinary Sector: 
MOLECULAR BIOLOGY
Semester: 
Second semester
Year of study: 
1
Language of instruction: 

Italian and English

Learning outcomes of the course unit

KNOWLEDGE AND UNDERSTANDING
The main goal of the course is to provide students with the tools necessary for a detailed and critique analysis of the structure of proteins and their macromolecular complexes. The first part of the course is dedicated to the understanding of the physical-chemical properties of the amino acids and their interaction within a protein. During the second part of the course, students are challenged with practical exercises on the structural analysis of protein models by means of open source software.

APPLYING KNOWLEDGE AND UNDERSTANDING
The educational objective of the course is to achieve the necessary knowledge for a critical analysis of the structure of proteins and nucleic acids. By the end of the course students will have acquired the skills necessary to deal with the analysis and experimental study of biological macromolecules. They will learn how to retrive protein and nucleic acid coordinates from the PDB database, recognize the fold and use sofware for a detailed analysis of their structure.

MAKING JUDGEMENTS
The course is aimed at increasing the ability to critically analyze the structure of proteins, nucleic acids and their interactions.

COMMUNICATION SKILLS
The course includes significant activity of classroom discussion aimed at developing the ability of students to transfer skills acquired in support of their arguments. In the final exam, students must take an oral presentation on the structure and function of an assigned protein.

LEARNING SKILLS
The many advancements of scientific research, particularly in the field of molecular biology require a continuous updating of skills. For this reason, the course aims to provide the necessary tools to achieve a wider knowledge and to align skills to the advancement in molecular biology research.

Course contents summary

The amino acids
Peptide bond
Secondary structure
Tertiary structure
Quaternary structure
Protein Folding
Enzymes
DNA-protein interaction
Membrane proteins
Fibrose proteins
Structure determination of proteins and nucleic acids by cryoEM

Course contents

Physico-chemical properties of amino acids, the peptide bond, phi and psi angle of rotation, the Ramachandran diagram.

Protein synthesis.

Secondary structures: Alpha helixes, 3.10 and Greek pi, beta sheets, loop regions.

Topological diagrams, calcium-binding helix-turn-helix motifs, beta hairpins, Greek a motif, beta-alpha-beta motif.

Alpha helix structures: inter-helix contacts and superstructural organization of alpha-helix proteins, four helix bundle, globin folding.

Alpha-beta structures: TIM barrel structure, Rossmann folding.

Beta structure: "barrels" formed by antiparallel beta strands; Greek key motif; "jelly roll" (vitamin A-binding proteins; neuraminidase; gamma-crystallin; immunoglobulin and immunoglobulin-like proteins.

Protein folding: conformational flexibility, thermodynamic and kinetic factors that affect folding, isomerization of proline residues, structure and function of GroEL/GroES chaperonines.

Proteins with enzyme activity: serin protease, enzyme-substrate complex, Km, Kcat, Vmax, the transition state, mechanism of action of chimotrypsin, specificity, convergent evolution.

Post-traslational modifications: phosphorylation, acetylation, n-linked glycosylation, methylation, ubiquitylation, palmitoylation.

DNA structure.

DNA recognition by prokaryotic transcription factors: the helix-turn-helix motif, specific and non-specific interactions, Cro, lambda repressor, Lac operon repressor, CAP, tryptophan repressor, allosteric effectors that alter the affinity of protein for DNA.

DNA recognition by eukaryotic transcription factors: TBP, specific sequence interactions, hydrophobics and plasticity of DNA, homeodomain proteins, POU regions. Zinc finger motifs, GCN4 leucin zipper.

Membrane proteins: bacteriorodopsin, porines, potassium channel, hydropathy graphs, Cys-loop ion channels.

F1F0 ATPase structure.

Fibrous proteins: alpha cheratines, collagen, fibroin.

AN INTRODUCTION TO ELECTRON CRYO MICROSCOPY FOR BIOLOGICAL MOLECULES

The aim of the lectures and workshop is to explore the principles of electron cryo microscopy and to understand how the method has grown over the decades to become such a powerfully effective tool for determining the three dimensional structures of biological macromolecules at high resolution. The course will cover topics including sample preparation, data collection and processing, three dimensional image reconstruction, and model generation and validation. We will also discuss developments of electron microscopy, including applications in drug discovery and tomography. We will go through a tutorial to prepare a three dimensional map from experimental data.

I. Background to transmission electron microscopy and image formation.
Why choose electrons? Comparison of interactions of X-ray and electrons with biological molecules. Electron Optics. Amplitude and phase objects. Elastic scatter versus inelastic and radiation damage. Phase contrast in cryo-TEM image formation. Contrast transfer and Contrast transfer function (CTF) correction.

II. Methods to prepare grids for TEM
Sample preparations for TEM: Negative-stained EM and cryo-EM. Sample delivery methods.

III. Basic theory for 3D reconstitutions
3D reconstruction from 2D projections.

IV. Cryo-EM data collection, data processing, map validation and methods for model building and refinement
Optimization of signal on the detector: principles of direct electron detectors. The electron microscope as an interferometer: coherence and defocus – why these are important to achieve maximum resolution. Optimization of dose to minimize sample damage.
Will cryoEM become cheaper and more democratic? Development of economic detectors and 100 keV machines.
Maximum-likelihood (ML) for image alignment and classification (RELION). Beam induced movement and correction. Map validation: Fourier shell correlation (FSC).
Model building and refinement. Focused classification. 3D variation analysis.

V. The future of cell biology: cryo-tomography
Preparing samples for tomography by milling. Specimen tilting, energy filtering. Sub-tomogram averaging. Correlative microscopy.

Resources:

Videos

An excellent general overview by Yifan Cheng, UCSF https://www.ibiology.org/ibioseminars/single-particle-cryo-em.html
“Getting Started in Cryo-EM” with Professor Grant Jensen at the California Institute of Technology. Video lectures recorded by Prof Grant Jensen, available on YouTube: http://cryo-em-course.caltech.edu/

Excellent introductory lecture series on cryoEM at the MRC LMB, 2017

https://www2.mrc-lmb.cam.ac.uk/research/scientific-training/electron- microscopy/

Publications

Methods in Enzymology vol 579, The resolution revolution: recent advances in cryoEM, edited by Tony Crowther.

Henderson R. Realizing the potential of electron cryo-microscopy. Q Rev Biophys. 2004 37:3-13.

Scheres, S.H.W. A Bayesian view on cryo-EM structure determination. 2012. Journal Molecular Biology 415, 406-418.

Baker and Henderson (2012), Int. Tables for Cryst., Vol. F, p. 451-463. https://bakerlab.ucsd.edu/publication-pdfs/2012-Baker-Henderson_IT- F_593.pdf

Passmore, L.A. and Russo, J.C. Specimen preparation for high-resolution cryo- EM. Methods Enzymol. 2016; 579: 51–86. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5140023/

Frank, J. (2006) Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State, OUP

Recommended readings

Branden C., Tooze J. INTRODUZIONE ALLA STRUTTURA DELLE PROTEINE (Zanichelli, II Ed., 2001)

Petsko, G.A., Ringe D., STRUTTURA E FUNZIONE DELLE PROTEINE (Zanichelli, 2006).

Nelson D.L., Cox M.M. I PRINCIPI DI BIOCHIMICA DI LEHNINGER (Zanichelli, III ed., 2002)

David Whitford, PROTEINS STRUCTURE AND FUNCTION (Wiley)
http://books.google.it/books?id=qbHLkxbXY4YC

Teaching methods

The course consists of in classroom lessons in which the main topics covered by the program will be presented and a theoretical and practical part with computer exercises in which students will learn to determine and analyze the structure of proteins and nucleic acids using CryoEM. In addition to the textbooks, students have access to teaching material on the course website, including the slides used in lectures and scientific articles made available by the teacher.

Assessment methods and criteria

For the current academic year, it is expected that the knowledge acquired, as well as the ability to use it in practice, will be verified with a written and oral exam in which the students will be invited to describe in-depth specific topics covered in the course and to demonstrate the skills acquired in relation to the theoretical and technical part regarding the structural determination of proteins and nucleic acids by CryoEM (which will serve to evaluate the knowledge acquired and understanding of the subject). This will serve to assess the ability to apply knowledge and understanding, as well as the autonomy of judgment and the ability to communicate ideas and concepts with clarity and properties of language.
The final grade will be derived from a balanced evaluation of the various aspects mentioned. Honors will be awarded in the case of students who show excellent knowledge and understanding of the subject, excellent ability to apply them, good judgment autonomy, and mastery of the disciplinary lexicon.

Other informations

Class schedule, exams dates, slides and other teaching resources can be found at the url:
https://elly2021.scvsa.unipr.it/course/view.php?id=225