Rationale & Goals: In this day of exploding bioinformatics information from genomics and proteomics, it is ever more important to be conversant with macromolecular three-dimensional structure, and how it relates to protein and nucleic acid function and drug design. This workshop will enable participants to find published macromolecular structure data, and visualize and interpret 3D macromolecular structure. Participants will be enabled to incorporate computer visualization and qualitative analysis of 3D structure of protein, DNA, RNA, and protein-ligand interactions into their teaching and research. Those who wish can prepare interactive macromolecular structure presentations, such as those at MolSlides.Org.

Software: The central tools for this workshop (all free) are:

FirstGlance in Jmol (firstglance.jmol.org) and Jmol java applet	Operates in all popular browsers and platforms, including Windows, OSX, and linux. Operates on-line only. Open source.	Assumes that you are already familiar with principles of protein structure. Adopted by Nature Structural and Molecular Biology and other bioinformatics resources.	Built with the free, open source Jmol java applet.
Protein Explorer in Chime (www.proteinexplorer.org). and Chime plugin	Operation limited to Windows or Mac Classic. Operates on-line or off-line (downloaded). Open source.	Much more powerful than FirstGlance. Extensive help and reference materials useful to both students and researchers. Much easier to use, yet much more powerful than RasMol. Won the 2003 MERLOT Classic Award in Biology for exemplary online learning resources: "The Protein Explorer has revolutionized the teaching of biology at a molecular level". Able to build MolSlides. Adopted by numerous bioinformatics resources.	Built with the free MDL Chime plugin (source code not open).

Level & Pace: This workshop is designed for graduate students and researchers familiar with basic biochemistry, but with no previous molecular visualization software experience. It progresses rapidly to powerful tools that will be of interest to specialists in protein structure and bioinformatics. Experienced participants are encouraged to work at their own speed, ahead of the group -- there is plenty of power to discover within Protein Explorer and its connections to complementary structural bioinformatics resources!

Day 1 Morning 10:40-12:10, Monday June 26. Seminar Room.     Review of Protein Chemistry and Structure.     Introduction to Structural Bioinformatics.

Review of Protein Chemistry and Structure.
1. Central Dogma: DNA mRNA Protein.
2. 20 Amino acids
   • Codon = 3 nucleotides; 4 nucleotides³ = 64 codons.
   • 3 letter and 1 letter codes.
   • Chemical properties (Structures in Chime: Side-by-Side - All-At-Once - Double-Bonds-pK_R - Properties / Venn Diagram)
     • Polar, uncharged (cf. water)
     • Charged
     • Hydrophobic (aliphatic vs. aromatic)
3. Polypeptide chain geometry and steric restrictions
   • Planarity of the Peptide Bond
   • Phi/Psi angles and steric collisions (QuickTime Movie)
   • Ramachandran Plots: MolProbity, Side-by-side
   • Pro (helix breaking), Gly (turns)
4. Covalent and non-covalent chemical bonds
   • Covalent bonds: lengths and angles nearly constant
   • Non-covalent bonds: variable lengths and angles
     • Salt bridges (up to 4.0 Å in proteins)
     • Hydrogen bonds (2.5-3.5 Å in proteins)
     • Cation-pi orbital interactions (up to 6.0 Å in proteins)
     • van der Waals interactions (up to 4.5 Å in proteins)
5. Secondary Structure
   • Primary, Secondary, Tertiary, Quaternary Protein Structure
   • Alpha helices (Requires a Gecko browser: Helix in Chime*)
   • Beta strands and sheets (Requires a Gecko browser: Sheet in Chime*)
     *Thanks to moleculesinmotion.com
   • Turns (Glycine)
6. Folding: hydrophobic collapse
7. Protein folds cannot be reliably predicted from theory
   
   
8. Introduction to Structural Bioinformatics
   • Why do we care about 3D macromolecular structure?
   • What are 3D structure data?
   • Where do 3D structure data come from?
   • How much 3D structure knowledge do we have?
   • Primary and Derived 3D Structure Databases

Day 1 Afternoon 14:40-17:50, Monday June 26. Computer Lab.     FirstGlance in Jmol for basic macromolecular visualization.     Finding published molecules of interest.

This afternoon, work in linux, in Mozilla or Firefox.


9. At firstglance.jmol.org, enter 1PGB (a Protein Data Bank identification code). Try these controls:
   1. Introduction
   2. and
   3. Top 2 rows of views
   4. Vines
   5. Buttons
   6. Center Atom
   7. Reset
   8. More Views
      • Cation-pi interactions and salt bridges (1AXI, 1M4U PQS for inter-chain)
      • Distances
      • Color by uncertainty
10. Explore 1HHO in firstglance.jmol.org, especially:
    1. Ligands button
    2. for full names of ligands
    3. Hide
    4. Find (explain the distributions of gly, pro, ala, glu, phe, viewed one at a time)
    5. Contacts to HEM
    6. 
11. 1BKX: More Views, Non-standard amino acids
12. 2ACE:
    
    
13. Finding published molecules of interest:
    Browsing
    1. Atlas of MacroMolecules: www.umass.edu/microbio/chime/pe_alpha/atlas/atlas.htm
    2. PDB at a Glance: cmm.info.nih.gov/modeling/pdb_at_a_glance.html
       
       Searching
    3. PDB Lite: www.pdblite.org
    4. Advanced search engines at the World Wide Protein Data Bank (wwpdb.org)
       • RCSB (USA) pdb.org (click Advanced Search)
       • PDB-Japan XML-Search
    5. OCA (Israel) http://bioportal.weizmann.ac.il/oca-bin/ocamain

Day 2, 13:00-17:50 Tuesday June 27. Computer Lab.     Protein Explorer.     Saving MolSlides.     Exploring Your Molecule.

This afternoon, work in Windows ME (virtual machine under linux) for compatibility with Chime and Protein Explorer.
To use Protein Explorer outside the Engineering School teaching computer laboratory:
• Protein Explorer works only on Windows or Mac PPC OS9 (Classic). (On Intel Macs, use Windows -- see parallels.com)
• Download and install the MDL Chime browser plugin.
• Mac PPC only: Download and install Netscape 4.8.

Use Firefox (or Netscape 7.2 or Mozilla).
• Internet Explorer cannot save MolSlides, and usually cannot display the Features of the Molecule control panel in Protein Explorer.
• Netscape 4.8 cannot save MolSlides, but does everything else in Protein Explorer, and it works with older Chime-based websites.
• Netscape 8 does not work with Protein Explorer.
Go to www.proteinexplorer.org to use the latest version, PE 2.78 Alpha (June, 2006).
Skip the PE Demo Movies -- use them for review (if you haven't used PE for a few months) or to start friends who didn't attend this course.

PE: FirstView

14. Click Quick-Start ... to display Gal4:DNA.
15. Organization of PE into 3 frames: control panel, molecular image, and messages.
16. Use the mouse to rotate the molecule; click to identify atoms.

    PE: Features of the Molecule

17. Understanding and using information provided in the PDB file header by the authors of the structure.
18. Enter 1E3Q in slot at FrontDoor (it has all Features).
19. The Help/Index/Glossary (green for "go"), a major component of PE's knowledge base.

20. Undo, History

    Saving MolSlides

21. Detailed Procedure for Saving MolSlides
22. Save This View, Add a MolSlide
23. MolSlide Manager, taking notes in MolSlides
24. Exporting & Saving MolSlides to your disk
25. Viewing MolSlides

    PE: QuickViews

26. Selecting, emphasizing, and hiding portions of the molecule.
27. Selecting arbitrary atoms/chains/residues by clicking on them.
28. Saving/recalling selected sets.
29. Zooming, centering.
30. Backbone, trace, cartoon, stick, ball and stick, spacefill to van der Waals radii.
31. Coloring by element (Corey, Pauling, Koltun color scheme).
32. Coloring cartoons by secondary structure.
33. Identifying the amino and carboxy termini (5', 3' ends): N->C Rainbow (Group) color scheme.
34. Interpreting the distribution of hydrophobic, polar, and charged residues (Polarity color schemes).
    1. Potassium channel: 1bl8. Trp prefers lipid-water interface.
    2. Gramicidin in a lipid bilayer: bilagram.pdb
35. Coloring to distinguish A, T, G, C, U. How to distinguish DNA from RNA. (Cf. 104d)
36. Coloring by disorder: temperature factor coloring.   Thermal vs. static disorder.

37. PE Site Map

38. Explore Your Molecule
    • Answer these questions about your molecule: Discovery in Protein Explorer (feel free to use FirstGlance also)

Residue sequence ranges for the CDR's in the Fab of 1FDL are: Heavy chain (H) CDR1: 31-35 CDR2: 50-66 CDR3: 98-105 Light chain (L) CDR1: 24-34 CDR2: 50-56 CDR3: 90-97 For shortcuts and tricks in using PE to visualize epitope-paratope contacts, see step #35 in this Antibody Structure Tutorial.

Sequences

39. Protein Explorer's clickable Seq3D
    1. Sequence to 3D structure mapping.
    2. Finding all instances of one amino acid (e.g. cysteine).
    3. Selecting and coloring an arbitrary range of residues (see example in box at right).

40. PE's MSA3D (via PE Site Map, Advanced Explorer): Introduction to multiple sequence alignments (for ConSurf).

41. External Resources (via PE Site Map): Structural Bioinformatics Servers that complement PE.
    1. ConSurf: regions conserved or hypermutable in evolution
    2. MolProbity: all-atom contact analysis -- add hydrogens, then
       • See and correct Asn, Gln, His side-chain flips
       • See atomic clashes and evaluate overall clash score (1cbx)

42. Importing and Applying MolSlides: Saving a PE Session.
    • Procedure

43. Multiple-Model NMR Results (1JSA, 1CFC)
    1. Most representative model (via PE Site Map -> External Resources).
    2. NMR Control Panel.
    3. Animation simulates thermal motion (Click "Animations" at the FrontDoor).

44. Noncovalent Bonds: Contact-Decorated Surfaces. Example: Gal4 contacting DNA (1d66), showing:
    1. Sequence specific recognition DNA bases by zinc finger domain of protein
    2. Hydrophobic protein-protein interaction
    3. Nonspecific charge interactions at DNA backbone phosphates

45. Preferences in Protein Explorer (beneath the message box).
46. Aliases for RasMol/Chime commands (beneath the message box).

47. QuickViews Boolean (scroll down in the QuickViews control panel).
    1. Example: In 1FDL, display Fab atoms contacting lysozyme, then overlay (DISPLAY) a cartoon display of all protein. Color the cartoon by Chain, then by N->C Rainbow, then by Structure.

48. Challenges. (The challenge questions will be given during class. They are not on-line.)
    
    
49. Structural Genomics: Worldwide Protein 3D Structure Knowledge
    1. How are 3D macromolecular structures obtained? Crystallography, NMR, and homology modeling.
    2. What fraction of the human proteome has known structure? A few percent.
    3. Is Structural Genomics the answer? Not in the next few years.
    4. Intrinsicially unstructured proteins:
       About 10% of proteins are thought to be fully disordered to support their functions, and 40% of eukaryotic proteins have at least one long disordered region. Examples.

50. Searching by structure without reference to sequence: (Try the bacterial cell division protein 1FSZ^§.)
    Structure is more conserved than sequence! (Chothia et al., 2003; Precis)
    1. Shindyalov & Bourne's Combinatorial Extension cl.sdsc.edu/ce.html
    2. NCBI's Vector Alignment Search Tool (VAST) www.ncbi.nlm.nih.gov/Structure/VAST/vast.shtml

    Model of SV40 Capsid showing icosahedron.

51. Specific Oligomers vs. Crystal Contacts
    • Specific oligomers (via PQS): 1K28, 1IO1, 1M4U (hydrophobic patch covered in PQS), virus capsids.
    • Crystal Contacts (4MDH via PE's Site Map, External Resources).

52. Animations & Morphing (Click "Animations" at PE's FrontDoor).
    1. Animation of NMR ensembles of models to simulate thermal motion
    2. Animation of ligand binding (HIV protease inhibitor; protein bending DNA)
    3. Morphing conformational changes
       • Rationale: Protein Morpher (requires Netscape 4)
       • Flagellar Hook

Day 5, 13:00-17:50 Friday June 30 - Seminar Room.:     Educators' Resources.     Molecular Modeling.

Again, today we will spend a large amount of time answering questions ("challenges") about structures. Use either FirstGlance in Jmol or Protein Explorer, whichever gives the answer more easily.

51. Challenges.
    • Bacterial Flagellar Structures

    For Educators

52. Lesson Plans (at PE's FrontDoor)

53. About Protein Structure (at PE's FrontDoor)

54. World Index of Molecular Visualization Resources molvisindex.org
    1. Hundreds of Chime-based tutorials indexed by macromolecule
    2. Chime-based resources en Español
    3. Sources of atomic coordinate (PDB) files (metabolites, inorganic crystals, lipid micelles, etc.)
    4. Galleries, Molecular Sculpture and Physical Models, Software

55. MolViz.Org www.umass.edu/microbio/chime
    1. BioMolecular Explorer 3D (for students ages 15-19). Soon to be available on CD with Chime and Netscape 4.8 installers.
    2. Amino Acid Quizzer
    3. DNA, Hemoglobin, Antibody, MHC
    4. Lipid Bilayers and Gramicidin Channel
    5. IR Spectra with animated vibrations
    6. Toobers in Science Education
    7. History of Visualization of Biological Macromolecules
       Where did Chime come from? What about Fred's Folly and Byron's Bender? See early computer images, physical models including the latest by computer-driven laser-powered rapid-prototype engineering, and the latest molecular sculpture.
    8. Knots in Proteins

56. Building a web page with hyperlinks to Protein Explorer that prespecify molecules for your teaching or research. Examples.   Methods.   Detailed methods.
    
    

    ---

57. Modeling vs. Visualization
    
    Optional Topics by Participants' Request -- Time Permitting (or Later Private Sessions):

58. Homology (comparative) modeling: Introduction.

59. Aligning two or more chains or molecules, and how to view the alignment.
    1. The CE site cl.sdsc.edu/ce.html will align any two protein chains quickly and easily (but hetero atoms are discarded).
    2. DeepView www.expasy.ch/spdbv/mainpage.html can align anything (one or more than one chains), selecting any subset of atoms for the alignment (other atoms following), and retaining hetero atoms. The results can be saved as a PDB file, but will need manual editing to separate models with MODEL [N] and ENDMDL records so that Protein Explorer can distinguish the models. Gale Rhodes provides a DeepView tutorial: click on the section Comparing Proteins.

60. Mutating your model:
    1. Changing residue sidechains and rotamer minimization with DeepView
    2. DeepView beginners should start with the superb Molecular Modeling for Beginners by Gale Rhodes, Univ. Southern Maine.
    3. DeepView resources are indexed at molvisindex.org.

• Molvis-list, jmol-users, PDB-list, etc. available from molvisindex.org.
• Yours truly: emartz@microbio.umass.edu (But please direct questions about RasMol or Chime or Protein Explorer to the molvis-list list, first item above.)