Bits protein structure
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This document provides an overview of protein structure analysis tools and techniques: 1) It describes exploring the Protein Data Bank (PDB) to view and analyze X-ray crystallography and NMR protein structures, comparing similar structures, and using tools like FoldX for in silico mutagenesis and homology modeling. 2) Key concepts covered include PDB file formats, atomic coordinates, B-factors, resolution, RMSD, and the principles of X-ray crystallography, NMR structure determination, and homology modeling. 3) Visualization software like YASARA, SwissPDBViewer and PyMOL are introduced for viewing protein structures from the PDB.
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Bits protein structure
- 1. BITS Training Protein Structure Joost Van Durme VIB Switch Laboratory Vrije Universiteit Brussel http://www.bits.vib.be/training
- 8. Human vs parasite Parasite Active site
- 9. 1918 Influenza Epidemic Influenza Virus
- 10. NEURAMIDASE POCKET SIALIC ACID
- 12. RELENZA TAMIFLU 5.000.000+ doses in NL
- 13. Trouw – 3 maart 2009
- 14. RELENZA TAMIFLU WT K i = 1.0 H274Y K i = 1.9 WT K i = 1.0 H274Y K i =265 H274Y H274Y
- 16. Principle of X-Ray crystallography initial model electron densities
- 17. X-Ray structure
- 19. Occupancy ATOM 625 C ILE A 77 -11.322 28.374 -1.179 1.00 28.77 C ATOM 626 O ILE A 77 -11.946 29.453 -1.112 1.00 28.84 O ATOM 627 CA AILE A 77 -11.432 27.329 -0.087 0.70 28.15 C ATOM 628 CB AILE A 77 -12.918 26.874 0.087 0.70 28.64 C ATOM 629 CG1AILE A 77 -13.042 25.758 1.141 0.70 26.75 C ATOM 630 CG2AILE A 77 -13.516 26.421 -1.241 0.70 28.13 C ATOM 631 CD1AILE A 77 -13.378 26.302 2.501 0.70 26.47 C ATOM 632 CA BILE A 77 -11.423 27.327 -0.082 0.30 28.50 C ATOM 633 CB BILE A 77 -12.874 26.775 0.117 0.30 28.79 C ATOM 634 CG1BILE A 77 -13.519 26.423 -1.227 0.30 28.62 C ATOM 635 CG2BILE A 77 -13.748 27.739 0.916 0.30 28.40 C ATOM 636 CD1BILE A 77 -14.720 25.518 -1.100 0.30 28.69 C ATOM 637 N ARG A 78 -10.521 28.048 -2.183 1.00 28.70 N ATOM 638 CA ARG A 78 -10.258 28.952 -3.268 1.00 28.47 C ATOM 639 C ARG A 78 -10.857 28.469 -4.584 1.00 28.22 C 2VWC
- 21. B-factor www.YASARA.org Low High
- 28. Structural data is stored in the Protein Data Bank (PDB) http://www.pdb.org Protein Data Bank (PDB)
- 32. ©CMBI 2009 PDB important records (3) and at the end of the PDB file the “real” data: ATOM one line for each atom with its unique name and its x,y,z coordinates ATOM 1 N THR 1 17.047 14.099 3.625 1.00 13.79 1CRN 70 ATOM 2 CA THR 1 16.967 12.784 4.338 1.00 10.80 1CRN 71 ATOM 3 C THR 1 15.685 12.755 5.133 1.00 9.19 1CRN 72 ATOM 4 O THR 1 15.268 13.825 5.594 1.00 9.85 1CRN 73 ATOM 5 CB THR 1 18.170 12.703 5.337 1.00 13.02 1CRN 74 ATOM 6 OG1 THR 1 19.334 12.829 4.463 1.00 15.06 1CRN 75 ATOM 7 CG2 THR 1 18.150 11.546 6.304 1.00 14.23 1CRN 76 ATOM 8 N THR 2 15.115 11.555 5.265 1.00 7.81 1CRN 77 ATOM 9 CA THR 2 13.856 11.469 6.066 1.00 8.31 1CRN 78 ATOM 10 C THR 2 14.164 10.785 7.379 1.00 5.80 1CRN 79 ATOM 11 O THR 2 14.993 9.862 7.443 1.00 6.94 1CRN 80
- 33. PDB entry
- 34. PDB entry
- 35. PDB entry
- 37. YASARA View nomenclature Atom Residue = any continuous stretch of atoms sharing the same residue name, residue number and molecule name Molecule = any continuous stretch of residues sharing the same molecule name (PDB calls this a CHAIN) Object = a collection of molecules and additional items
- 39. Atom nomenclature N-term C-term C α C β C γ O γ N N O C α C β C C C γ C δ 1 C δ 2 OT1 OT2
- 47. FoldX plugin for YASARA
Editor's Notes
- Also drugs against invaders: bacteria, viruses.... Trypanosoma (sleeping disease): eukaryotic lifeform. So many proteins, just like us. So if we make a medicine that eg knocks out the glycolysis of trypanosomas, we should be very careful that we don’t knock out the glycolysis of the host. So we have to study differences in proteins between tryp and host. In picture you see active site of trypanosomas. Then we can design a ligand that blocks the trypanosomas protein active site but not that of the human.
- Neuraminidase inhibitors Neuraminidase helps to release the viral particle from the infected cell so it can infect another cell. It breaks sialic acid bonds in the membrane so that a membrane vesicle with virus inside can travel to another cell. So the neuraminidase binds sialic acid.
- Smart as we are, we made molecules that look like sialic acid (relenza and tamiflu) to neutralize the neuraminidase and prevent the virus from being mobile.
- But at some point the tamiflu lost its action against neuraminidase.
- On binding tamiflu, the conformation of the Glu 277 side chain of the wt enzyme is altered such that it exposes a hydrophobic site with which the pentyloxy group of oseltamivir interacts. In the mutant enzyme, the bulkier Tyr residue at 275 displaces the carboxyl group of Glu 277 into the binding site, such that it disrupts the hydrophobic pocket and causes a change in conformation of the pentyloxy substituent of oseltamivir (Fig. 1a), with consequent reduction in affinity of binding of some 300-fold or greater. Luckily for us, relenza’s side group isn’t that big and still fits in the pocket of the mutant neuraminidase. The question is, what will we do if the virus also beats relenza?
- Variations in time: x-ray experiment takes seconds. Motions happen in femtoseconds. So motions can be the source of uncertainty. In space: different molecules in the crystal have a different conformation of a sidechain
- The resolution for a given crystal depends on the ordering of the molecules in the crystal. That is, how close the unit cells thoughout the crystal are to being identical copies of one another. Rule: the larger the molecule, the lower the resolution of the data. At low resolution (4A): overall shape of molecule, no interactions! 3A: path of chain can be traced 2A: sidechains become visible 1.2-0.9A: hydrogen atoms become visible, occupancies easily detectable These structures require fewer geometric constraints during refinement and give a better indication of the true geometry of protein structures.
- X-rays are scattered through the crystal generating a diffraction pattern from which an initial model of the structure is generated based on the electron density map. This model can also be a homology model of the new structure based on a close homolog of which we know it will adopt more or less the same structure. Using scattering theory it is possible to calculate computationally the expected diffraction pattern. Usually this will differ from the observed one. Refinement involves iteratively modifying the model untill the computed diffraction pattern has a best fit with the observed diffraction pattern. Ferredoxin was incorrectly solved due to wrong space group assignment, but all the water molecules made up for it. 344 waters on 117 residues. Refinable parameters The structural model describes a collection of scattering centres (atoms), each located at a fixed position in the crystal lattice, and with some degree of mobility or extension around that locus. In adjusting the structural model to improve the fit between calculated and observed diffraction patterns, the crystallographer may vary these and other parameters. Refinable parameters are those that may be varied in order to improve the fit. Usually they comprise atomic coordinates, atomic displacement parameters, a scale factor to bring the observed and calculated amplitudes or intensities to the same scale. They may also include extinction parameters, occupancy factors, twin component fractions, and even the assigned space group. Relations between the refinable parameters may be expressed as constraints or restraints that modify the function to be minimized.
- Deposition procedure: an experimentalist choosing the best 20 structures from a much larger ensemble can result in very misleading statistics! eg. the best 20 models may be the best solution with only small variations, so RMSD will be small but further down the original list are alternative solutions, which are less consistent with the data but radically different!