Structural Biochemistry
Code
7637
Academic unit
Faculdade de Ciências e Tecnologia
Department
Departamento de Química
Credits
6.0
Teacher in charge
Maria dos Anjos Lopes de Macedo, Maria João Lobo de Reis Madeira Crispim Romão
Weekly hours
4
Teaching language
Português
Subject matter
Part I
- Basic Concepts in NMR (review); 1D and 2D spectra (homo-and hetero-nuclear): applications to proteins
- Identification and assignment of signals in protein 2D spectra; secondary structural elements determination based on chemical shifts
- Backbone assignment of proteins using triple resonance experiments - methodology
- Determination of protein structures in solution; NMR constraints and methodology for structure calculations
- Relaxation and protein dynamics
- NOE effect and Saturation Transfer Difference experiments: application to protein - ligand interactions
- NH exchange and protein structural information.
Part II
- Introduction to protein crystallography; review
- Crystallization of proteins; Lattice, Symmetry and Space Groups
- X-ray sources and detectors; diffraction; reciprocal space
- Diffraction and Fourier transform; The Phase Problem and how to solve it.
- Model building and Refinement; Methods of Validation
- Molecular Electron Microscopy
- Other complementary Techniques: SAXS, ITC, DLS
Practical Classes and Seminars
NMR solution structure
- P1 : The NMR spectrometer ; 2D heteronuclear experiments, aquisition and procesing (TOPSPIN).
- P2 : Resonance assignment using 3D spectra. Introduction to BMRB - Biological Magnetic Resonance Data Bank.
- P3 : Solution structures determination using Cyana software - input data preparation, output data (violations detection & iterative process) and validation results (online tools). NMR vs X-ray structures.
- P4 : Protein ligand interactions; titrations followed by HSQC and data obtained by STD (eg CBM11 )
- S : Case study Oral presentation and discussion
X-Ray Crystallography
- P1: databases, PDB revisited
- P2: Preparation and optimization of protein crystals (Q1)
- P3: The X-ray diffractometer and diffraction experiment. Processing and analysis of data.
- P4: Solving a structure by MR method (Q2)
- P5: Analysis of electron density and model building; Model quality and validation criteria. (Q3)
- S: Seminars – Case study : Oral presentation and discussion
Bibliography
- "Structural Biology, Practical NMR applications", Quincy Teng, Springer (2005)
- “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)
- Cavanagh, J., Fairbrother, W.J., Palmer III, A.G., Skelton, N.J. ‘Protein NMR Spectroscopy. Principles and Practice’, Elsevier Academic Press (2007)
- Levitt, M.H. “Spin Dynamics. Basics of Nuclear Magnetic Resonance”, John Wiley & Sons, Ltd, England (2008)
- Relevant publications (reviews and articles) in more recent topics like protein relaxation and dynamics, methods and criteria of NMR structure calculation and validation, structural information obtained by SAXS ande cryo-EM.