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Semelhante a Class powerpoint.ppt
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Class powerpoint.ppt
- 1. Learning Objectives • Review the basics of protein structures • Know how to predict secondary structures • Appreciate both the potentials and limitations of 3D analysis • Predicting secondary structures • Guessing the 3D structure of your sequence • Further applications of 3D analysis
- 2. Primary, Secondary and Tertiary Structures • Proteins are made of 20 amino acids • Protein structure has 3 levels: – The primary structure is the sequence of a protein – The secondary structure is the local structure – The tertiary structure is the exact position of each atom on a 3D model
- 3. Primary Structures • www.expasy.org https://web.expasy.org/protparam/ • Swiss EMBnet in www.ch.embnet.org • And ….
- 4. Primary Structures • http://web.expasy.org/protparam/ • Estimated half-life: (mammalian reticulocytes, in vitro). (yeast, in vivo). (Escherichia coli, in vivo). Instability index: 40This classifies the protein as stable. Aliphatic index: 84.17 Grand average of hydropathicity (GRAVY): -0.275
- 6. Secondary Structures • Helix – Amino acid that twists like a spring • Beta strand or extended – Amino acid forms a line without twisting • Random coils – Amino acid with a structure neither helical nor extended – Amino-acid loops are usually coils
- 7. Guessing the Secondary Structure of Your Protein • Secondary structure predictions are good • If your protein has enough homologues, expect 80% accuracy • The most accurate secondary structure prediction server is PSIPRED
- 9. •Input Sequence (Single sequence or Multiple Sequence alignments; as raw sequence or fasta format) •Submission Details
- 10. PSIPRED Output psipred@cs.ucl.ac.uk • Conf = Confidence – 9 is the best, 0 the worst • Pred = Every amino acid is assigned a letter: – C for coils – E for extended or beta-strand – H for helix
- 13. Predicting Other Secondary Features • It is also possible to predict these accurately: – Transmembrane segments – Solvent accessibility – Globularity – Coiled/coil regions • All these predictions have an expected accuracy higher than 70%
- 14. Scratch Protein Predictor • http://scratch.proteomics.ics.uci.edu/
- 15. Transmembrane Domains • Discovering a transmembrane domain tells you a lot about your protein • Many important receptors have 7 transmembrane domains • Transmembrane segments can be found using ProtScale • The most accurate predictions come from using TMHMM
- 18. Using TMHMM • TMHMM is the best method for predicting transmembrane domains • Its principle is very different from that of ProtScale • TMHMM output is a prediction
- 21. Predicting 3D Structures • Predicting 3D structures from sequences only is almost impossible • The only reliable way to establish the 3D structure of a protein is to make a real-world experiment in – X-ray crystallography – Nuclear magnetic resonance (NMR) • Structures established this way are conserved in the PDB database • “The PDB of my protein” is synonymous with “The structure of my protein”
- 22. Retrieving Protein Structures from PDB • All PDB entries are 4-letter words! – 1CRZ, 2BHL . . . • Sometimes the chain number is added: – 1CRZA, 1CRZB . . . • To access all PDB entries, go to www.rcsb.org – PDB contains 42,000 entries – PDB contains the structure of 16,000 unique proteins or RNAs • You can download the coordinates and display the structure
- 24. Displaying a PDB Structure • You can use any of the online viewers to display the structure • They will let you rotate the structure, zoom in and out, or color it • PDB files themselves are not human-readable
- 25. Predicting the Structure of Your Protein • The bad news: – It is very hard to predict protein 3D structures • The good news: – Similar proteins have similar structures • If your favorite protein has a homologue with a known structure . . . – You can do homology modeling • How? – Start with a BLAST (more about that in the next slide)
- 28. BLASTing PDB for Structures • If you get a very good hit, it means PDB contains a protein similar to yours • Your protein and this hit probably have the same structure
- 29. Be Careful! • Sometimes only one of the domains contained in your protein has been characterized • If that’s the case, the PDB will only contain this domain • Always check the alignments – Red line = full protein in PDB – Blue line = one domain only in this entry
- 30. Structures and Sequences • Highly conserved sequences are often important in the structure • Make a multiple-sequence alignment to identify these important positions • Highly conserved positions are either in the core or important for protein/protein interactions
- 33. 3D Predictions • If you want to predict the structure of your protein automatically, try the Swiss Model – Swiss Model makes the BLAST for you – The program does a bit of homology modeling – The process delivers a new PDB entry – You can access it at swissmodel.expasy.org • Swiss Model gives good results for proteins having homologues in PDB
- 39. 3D-BLAST • Use this technique if you have a structure and you want to find other similar structures • Use VAST or DALI to look for proteins having the same 3D shape as yours – www.eb.ac.uk/dali – www.ncbi.nlm.nih/vast
- 40. 3D Movements • Most proteins need to move to do their job • Predicting protein movement is possible using molecular dynamics – Check out this site: molmolvdb.mbb.yale.edu • Good molecular dynamics requires extremely powerful computers – Don’t expect miracles from standard online resources
- 41. Protein Interaction • Knowing “who interacts with whom” is one of the ultimate goals of predicting protein movement • These predictions are called docking analyses • Docking analyses are very difficult – Try www.biosolveit/FlexX/
- 42. Thanks for Your Attention