Protein Structure Visualization using

Transcription

1 Protein Structure Visualization using

2 Outline Protein Data Bank Protein structure visualization Homology modeling

3 Molecular Machinery: A Tour of the Protein Data Bank (mm.rcsb.org)

4 Protein Data Bank (PDB) 3D structural data of biological macromolecules Crystallography / NMR / electron microscopy / computation Freely accessible: Key resource for structural biology submission of structures to PDB required before publication Entries reviewed by PDB staff and automatically checked for plausibility

5 PDB holdings As of Sep. 21, 2016 Yearly Total

6 The PDB 1vh7 Search PDB ID Display seq or structure files in new tab Annotations PDB 101 Download

7 PDB file format (.pdb /.ent) A plain text file that includes atomic coordinates, observed side chain rotamers, secondary structure assignments, atomic connectivity,...

8 Protein Structure Visualization Tools RasMol Jmol UCSF Chimera PyMOL (Warren DeLano) VMD: Visual Molecular Dynamics In-browser viewers JSmol (JavaScript), PV (WebGL)

9 Homology modeling Sequence determines structure Sequence determines structure

10 Homology modeling Sequence Structure Homology Sequence Structure Homology modeling Construction of an atomic resolution model of a protein (query) from its amino acid sequence and an experimental 3D structure of a homologous protein (template). MODELLER Program for Comparative Protein Structure Modeling by Satisfaction of Spatial Restraints. Sali & Blundell (1993) Comparative protein modeling by satisfaction of spatial restraints. J. Mol. Biol. 234,

11 PyMOL simple visualization of structures analysis of topology, side chain packing, ligand interaction, etc superposition / structural alignment linking conservation with structure simple manipulations e.g. side chain mutagenesis publication quality figures function extension via plug-ins PyMOL wiki:

12 PyMOL GUI

13 PyMOL Basic commands fetch 4v32 # fetch & load 1THF from PDB hide everything show cartoon show sticks, hetatm spectrum count spectrum b # # # # # use menu to color by chain enable / disable objects show ligands as sticks Nt blue, Ct red color by b-factor color silver color red, ss h color blue, ss s # colors α helices red # colors β strands blue help cmd # get help

14 PyMOL - Alignment fetch 4v32 fetch 4v30 hide everything # show only single domain show cartoon, /4v32//A or /4v30//A # align 4v32 and 4v30 align /4v30//A, /4v32//A, object=aln # view sequence alignment set seq_view select ligands, hetatm and (not r. hoh) and (/4v32//A or /4v30//A) show sticks, ligands show spheres, ligands select binding_site, byres ligands around 4.0 show lines, binding_site color yellow, binding_site

15 Exercise superpose cereblon structures with different ligands Visualize cereblon from Magnetospirillum structures in PyMOL Get PDB IDs by searching PDB Align all of them onto 4v32 Visualize the ligands and their binding sites What's the common feature of the ligands? What's unique about the binding site

16 PyMOL - Visualizing sequence conservation Map conservation onto a structure by replacing the temperature factors (b-factor) with the conservation score 1) Download the sequence of 1VH7 from the PDB compare it to the.pdb file; what do you observe? 2) Perform a Protein BLAST search of 1VH7 (modified) sequence against nr70 3) Make an alignment using BLAMMER set Max identity to 0.8 and use Output format: ClustalW 4) Go to ConSurf at: Follow the instructions NOTE: Upload the MSA generated by blammer In the result page (e.g. ): Click Follow the instructions to produce a PyMol figure Click PDB_FILE to downloaded a revised PDB file. 5) Load the revised pdb into PyMOL and color it according to b-factors (1 var; 9 consv) load <PDB_FILE> spectrum b, cyan_meganta, minimum=1, maximum=9

17 PyMOL - Visualizing sequence conservation (rlt) Variable Average Conserved as cartoon show surface

18 Exercise Homology modeling using Modeller run HHpred on protein domain e.g. LRR domain of LRRK2 ( Tuesday exercise ) click on tab Create model pick sequences you want to use for homology modeling, e.g. Toll like receptor ( how would you cope with the gaps in the alignment? ) click Create model from manually selected sequences, an alignment will be generated submit job save the pdb file of the generated model and open it in PyMOL Highlight asparagines using command: color red, r. asn (illustrates their structural importance) you can check the model quality by clicking on the tabs (Toolkit)