Structural Biochemistry A
Objectives
This curricular unit will provide the students with theoretical and practical skills to:
- plan, execute and analyze protein crystallization assays;
- carry out a crystallographic characterization of the crystals obtained;
- collect and process diffraction data from a protein crystal;
- plan and execute the 3D structure determination of a protein;
- build, refine and critically analyze the 3D structural model of a protein;
- perform the structural analysis of the model obtained, compare it with models obtained by other methods and find similar structures in databases;
- carry out a basic interpretation of 1D and 2D NMR spectra of proteins;
- collect and process 1D and 2D NMR spectra of proteins;
- determine by NMR the structure of a protein with MW < 15 kDa.
General characterization
Code
11193
Credits
6.0
Responsible teacher
Pedro Manuel Henriques Marques Matias, Ricardo O. Louro
Hours
Weekly - 4
Total - 51
Teaching language
Português
Prerequisites
Available soon
Bibliography
- Structural Biology, Practical NMR applications, Quincy Teng, Springer (2005)
- H.J. Dyson, A.G. Palmer, “1.9 Introduction to Solution State NMR Spectroscopy”, Comprehensive Biophysics, 136-159, Edward H. Egelman, Eds, Elsevier (2012)
- “Introduction to Protein Structure” Branden, C.-I. & Tooze, J. Garland Pub. (1999)
- “Crystallography made Crystal Clear- A Guide for users of Macromolecular Models” G. Rhodes, 2nd Ed., Academic Press: San Diego, London (2000)
- Recent publications (articles and reviews) will be made available each year
Teaching method
- Theoretical classes - direct contact with the teaching staff in a classroom for presentation and discussion of the theoretical and theoretical-practical concepts included in the syllabus.
- Theoretical/Practical classes - direct contact with the teaching staff in classroom or laboratory for application of the concepts presented in the theoretical classes. The students will be divided into groups and each group will choose a project dealing with the structural characterization of a protein by X-ray crystallography and/or NMR. The work progress will follow as closely as possible the contents of the theoretical classes to allow the students a better assimilation of the concepts presented.
- Independent study - time used by the students to prepare project reports and study for the final examination.
Evaluation method
Evaluation - will consist of a written examination (45% of the final grade) and an oral presentation and discussion of the project(s) executed by the student (55% of the final grade).
Subject matter
- Crystal symmetry; crystallization methods; characterization of crystals;
- sources of X-rays, diffraction by single crystals, instruments and methods for diffraction data collection;
- The structure factor, electron density maps, the "phase problem" and methods for its solution;
- Methods for building and refining a crystallographic structural model; convergence criteria;
- Electron Crystallography and Electron Microscopy for 3D structure analysis;
- Validation methods for crystallographic protein 3D structures; structural comparison; crystallographic databases. Comparison with other methods of 3D structural analysis. On-line computational tools;
- Basic theory of 1D and 2D NMR. Structural information: angles, distances, chemical environment.
- Pulse sequences for data acquisition and spectral assignment. 2D and 3D methods.
- Protein structure determination methods by NMR: Proteins with MW <15kDa vs. proteins with MW >15kDa.