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 tool for this workshop is Protein Explorer (www.proteinexplorer.org). Protein Explorer is free, operates on Windows or Macintosh (also linux in a Windows subsystem), and is much easier to use, yet much more powerful than RasMol. Protein Explorer 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". Protein Explorer integrates several complementary bioinformatics servers, and has been adopted by numerous bioinformatics resources.

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, Monday August 1. Computer Lab. Basics. How to use Protein Explorer to visualize structural features of proteins. Saving MolSlides.

Use Firefox (or Netscape 7.2 or Mozilla); Internet Explorer is OK but usually cannot display the Features of the Molecule control panel in Protein Explorer. Netscape 4.8 works with most Chime-based resources and nearly all of PE but not with PE's MolSlides. Netscape 8 does not work with PE.
Go to the local copy of Protein Explorer, or to www.proteinexplorer.org
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.
Use PE 2.76 Alpha (July 24, 2005), not PE 2.45 Beta (January 2005).

PE: FirstView

1. Click Quick-Start ... to display Gal4:DNA.
2. Organization of PE into 3 frames: control panel, molecular image, and messages.
3. Use the mouse to rotate the molecule; click to identify atoms.
4. Identify and become familiar with the computer representations for chains, backbones, disulfide bonds, solvent, and ligands.

   PE: Features of the Molecule

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

8. Undo, History (new in PE 2.76 Alpha, June 2005)

   Saving MolSlides (new in PE 2.76 Alpha, June 2005)

9. Detailed Procedure for Saving MolSlides
10. Save This View, Add a MolSlide
11. MolSlide Manager, taking notes in MolSlides
12. Exporting & Saving MolSlides to your disk
13. Viewing MolSlides

    PE: QuickViews

14. Selecting, emphasizing, and hiding portions of the molecule.
15. Selecting arbitrary atoms/chains/residues by clicking on them.
16. Saving/recalling selected sets.
17. Zooming, centering.
18. Backbone, trace, cartoon, stick, ball and stick, spacefill to van der Waals radii.
19. Coloring by element (Corey, Pauling, Koltun color scheme).
20. Coloring cartoons by secondary structure.
21. Identifying the amino and carboxy termini (5', 3' ends): N->C Rainbow (Group) color scheme.
22. 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
23. Coloring to distinguish A, T, G, C, U. How to distinguish DNA from RNA. (Cf. 104d)
24. Coloring by disorder: temperature factor coloring.   Thermal vs. static disorder.

25. PE Site Map

Structural Bioinformatics

26. What are 3D structure data?
27. Where do 3D structure data come from?
28. How much 3D structure knowledge do we have?
29. Primary and derived 3D structure databases.

    Structural Genomics: Worldwide Protein 3D Structure Knowledge

30. How are 3D macromolecular structures obtained? Crystallography, NMR, and homology modeling.
31. What fraction of the human proteome has known structure? A few percent.
32. Is Structural Genomics the answer? Not in the next few years.
33. 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.

34. Importing and Applying MolSlides: Saving a PE Session.

35. Review of Hydrogen Bonds

36. Gaps in Amino Acid Sequences in PDB files.
    1. Insertions and non-physical gaps: 1igt.
    2. Physical gaps: 2ace, 1fod.
    3. Microheterogeneity: 1cbn.

37. 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. SearchFields at the Protein Data Bank www.pdb.org
    5. Prilusky's OCA http://bioportal.weizmann.ac.il/oca-bin/ocamain

38. Importing and Applying MolSlides: Saving/Restoring a PE Session.

39. 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

    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

40. 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).

    Model of SV40 Capsid showing icosahedron.

41. External Resources (via PE Site Map): Structural Bioinformatics Servers that complement PE.
    1. Probable Quaternary Structures: specific oligomers: 1k28, 1k93, virus capsids.
       vs. Crystal Contacts (4mdh).
    2. ConSurf: regions conserved or hypermutable in evolution
    3. 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)

Day 4 - Thursday August 4 - Computer Lab. Salt Bridges, Cation-Pi Orbital Interactions. Multiple Models (NMR). PE for Power Users.

42. Visualizing Cation-Pi interactions and Salt Bridges (QuickViews, DISPLAY; 1b07, 1axi)

43. 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.

44. 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).

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

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    Optional Topics by Participants' Request -- Time Permitting:

47. Homology (comparative) modeling: Introduction.

48. 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.

49. 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.

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

51. External Resources (via PE Site Map)
    1. Crystal Contacts
    2. Fewer or Single Chains
    3. Model Quality (& examples of errors in published PDB files)
    4. RCSB's Structure Explorer
    5. NCBI's Entrez Structure

    Advanced Explorer

52. The Noncovalent Bond Finder
53. Rolling probe surfaces and molecular electrostatic potential coloring
54. Including ligands in displays of cation-pi interactions and salt bridges

Day 5 - Friday August 5 - Seminar Room.: Animations and Morphing. Jmol. Resources for Educators.

55. 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

56. Presentations in Protein Explorer (PiPEs) (at PE's FrontDoor)

57. MolVis History and Future:
    1. Kinemages, KiNG
    2. RasMol
    3. DeepView
    4. Chime: Molvisindex.org, PE, Sting, MolUSC
    5. Future: Jmol (Chime-compatible applet & application, open-source)
    6. Exporting MolSlides to Jmol

    
    

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    MolVis Resources for Educators

58. Lesson Plans (at PE's FrontDoor)

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

60. 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

61. 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

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

• Molvis-list, 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.)
• Collaborations are invited that use Protein Explorer to display information about macromolecular structure, particularly information which may be the result of your research. The only example to date involving customization of PE is ConSurf. PE is used without modification by several other bioinformatics servers.