Syllabus
Practical Macromolecular 3D Structure Visualization & Structural Bioinformatics
A Two-Morning Workshop -- University of Massachusetts, Amherst

Group I: Monday June 3 & Wednesday June 5, 2019.
Group II: Monday June 24 & Wednesday June 26, 2019.
9:00 AM - 1:00 PM each day, Fine Arts Center 444.


Laptop Computers Welcome (bring your power cord).
FAC444 has Windows and macOS computers you may use.

Taught by Eric Martz, Ph.D.
principal author of FirstGlance in Jmol and MolviZ.Org,
team member of Proteopedia.Org, and coauthor of the ConSurf Server.


This document is on-line:
Workshops.MolviZ.Org

Please see the Workshop Announcement

Objectives: Participants will use highly user-friendly software for visual investigation of 3D molecular structures of proteins, nucleic acids, and their interactions with each other and with ligands, substrates, and drugs; and of protein evolutionary conservation. Hands-on experience will be largely with molecules of each participant's choosing. The software easily makes high quality molecular images and rotating molecules for Powerpoint slides.

Software: All software in this course is free, and works on Windows, Mac OS X, or linux. The major tool is FirstGlance.Jmol.Org, used >200 times/day worldwide, & adopted by several journals and resources.

Level & Pace: This workshop is designed for faculty, postdocs, research staff and graduate students 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 these tools!

        Get Started:

    Please get started on your own while the group gets settled:

  1. If you brought your own laptop, you are welcome use it. (iPads will be too slow.)
  2. Use the Chrome browser or Firefox or Opera. (Why?). If you do not have one of these, take a few minutes to install one (google.com/chrome, firefox.com).
  3. Go to our syllabus: Workshops.MolviZ.Org.
  4. Click on today's syllabus. Now you can see this document in your browser.
  5. Go to Atlas.MolviZ.Org.
  6. In the Atlas, choose any molecule deemed Straightforward and click on the link to FirstGlance. After a minute or so to load, you should see a rotating molecule. Have a look around at the information, views and tools in FirstGlance.
  7. If you have any difficulty or the molecule does not appear, or does not rotate, ask for help!


  8. Workshop Overview

  9. Workshop Overview (Powerpoint Slides)

  10. Optional take-home project: answer questions about your molecule and email your answers to Eric for feedback.
    1. Designed for undergraduates (11 questions):
    2. Designed for graduate students:
      • 18 Questions (Snapshot methods and #16-17 Polyview-3D are superceded by saving images/animations directly from FirstGlance)
      • Sample completed reports for a PDB code or a homology model (downloadable Powerpoint slides).
    The above questions can be used for assessment in a class you are teaching.
    Feel free to re-use/adapt any resources in this workshop!

  11. Nikhil Malvankar (Physics, UMass Amherst) took this workshop in 2012. Published his homology model in 2015.

  12. Protein Data Bank & PDB Codes
    Proteopedia.Org
    Crystallographic Resolution

  13. The Protein Data Bank (PDB) -- World Wide: -- USA:RCSB -- Japan:PDBj -- Europe:PDBe
  14. PDB identification code examples:



  15. Proteopedia.Org:
    1. Main page: green links connect text to molecular scenes.
    2. Molecules explained by users. Example:
    3. Explanations of structural biology terms and concepts, e.g. asymmetric unit, Protein Data Bank, hydrogen bonds, temperature value, and more: About Macromolecular Structure.
    4. Pages in Chinese, Japanese, Arabic, Turkish, Russian, etc. See anti-Alzheimer's drug in 14 languages.

  16. X-Ray Crystallography and Resolution
    X-ray crystallography generates an electron density map with resolution limited by the order in the protein crystal. The atomic model (shown as sticks below) must then be fitted into the map.
    (Technical: Endnote 1) (Technical: Endnote 2)
    C O N Electron Density Map
    (Technical: Endnote 3) Movie

  17. Electron Cryo-Microscopy is increasingly important

    Finding molecular models of interest
    Each participant should find a molecule of personal interest.

  18. Finding molecular models of interest:
    Each person: please find a 3D model of a molecule related to your research or interests.

    You will use the model that you select for the rest of the workshop.
      Ideal Model for Learning in This Workshop
    1. PDB code for an X-ray crystal structure with good resolution <= 3.0 Ångstroms.
    2. Has protein.
    3. Has ligand(s).
    4. Has a value for Rfree (free R).

      If An Empirical Model Of Your Protein Of Interest Is Not Available
    5. Consider using a homology model. See Limitations of Homology Modeling.
    6. Don't use a homology model if an empirical (X-ray or NMR) model exists for your protein. The empirical model will be more accurate. If unsure what to use, please ask the instructor.
    7. Use a homology model only if there are at least 120 amino acids in one chain forming a compact domain. Smaller homology models usually represent less than a single domain and are not useful. If unsure, please ask the instructor.

    How To Find Models:
    1. Go to UniProt.Org.
    2. Find your molecule. Ask for help if needed.
    3. Click on the blue Sequences button at the left.
    4. Make a note of the length of the full-length amino acid sequence.
    5. Click on the blue Structure button in the left column. (Examples: yeast gal4; human pla2g6; zebrafish acetylcholinesterase; human p53.)

    6. Follow instructions for the Simple search, and if necessary the Advanced search, at
      Is There An Empirical Model? ("Empirical" means determined by X-ray, NMR, or cryo-EM.)
    7. For any model that you find, pay attention to how much of the full-length sequence it covers.

    8. If there is no empirical model, is there a homology model?
      See How To Obtain Homology Models.
    9. For any model that you find, pay attention to how much of the full-length sequence it covers.
    10. To make your own homology model: (not necessary if you found an X-ray or NMR model!)
      1. Copy your amino acid sequence in FASTA format (e.g. from UniProt).
      2. Go to Swiss-Model.
      3. Click Create Account or Log in.
        It is worth bothering to create an account because it makes your jobs easy to find later. Without an account you might have to re-run the job and it can take over an hour.
      4. Click the blue Start Modeling button near the top center, or use the pull-down Modelling menu and click on myWorkspace.
      5. Paste your sequence in the large box.
      6. Click the large blue button Build Model.
    11. After Swiss-Model makes a model for you, download the model PDB file (see snapshot at right).
    12. Upload your homology model PDB file to FirstGlance:
    13. Use a homology model only if there are at least 120 amino acids in one chain forming a compact domain. Smaller homology models usually represent less than a single domain and are not useful. If unsure, please ask the instructor.

    14. Browsing Molecules: If you can't get a satisfactory model for your protein, look at one of these sites and pick the PDB code for another protein of interest.


    Review of Protein Chemistry and Structure.
    We'll quickly touch on just a few points here. Most of this section, and the links, are for participants who are educators.
    Standard amino acids.
    Click to see details.

  19. Central Dogma: DNA mRNA Protein.     DNA structure in Jmol / Estructura del ADN
  20. 20 Amino acids
  21. Polypeptide chain geometry and steric restrictions
  22. Covalent and non-covalent chemical bonds
  23. Typical hydrogen bond within a protein: hydrogen donor atom is covalently bonded to hydrogen; acceptor atom is not. In proteins, donor-acceptor distance can be 2.5 to 3.5 Å.
  24. Primary, Secondary, Tertiary, Quaternary Protein Structure
    Los cuatro niveles estructurales de las proteínas
  25. Secondary Structure
  26. Tertiary Structure: Folding & Hydrophobic Collapse
  27. Quaternary Structure
  28. Intrinsically Disordered Protein

  29. Evolutionary Conservation: ConSurfDB & ConSurf
      Enolase (4enl; a glycolytic enzyme) evolutionary conservation from ConSurf. Catalytic cleft is at the left. More..

  30. Identify Functional Sites In Your Molecule Using The ConSurf Server:
  31. Get your ConSurf calculation started while we discuss evolutionary conservation:

    1. Go to the ConSurf Server.
    2. Select Amino Acids.
    3. Check YES there is a known protein structure.
    4. Enter the PDB code, or upload your homology model.
    5. Select a chain for analysis. (If your PDB file has only one chain, it will be selected automatically.) ConSurf can analyze only one chain at a time.
    6. Check NO, you will not be uploading a multiple sequence alignment. (ConSurf will create one for you.)
    7. Leave all search parameters at their pre-set defaults.
    8. Select sequence homologs for ConSurf analysis:
      Check manually for a research-grade result.
      Check automatically for a quick but probably lower quality result.
    9. Leave the alignment method, calculation method, and evolutionary substitution model at their default settings.
    10. Enter your email address at the bottom of the form (this is important so you don't lose your results). Optionally enter a job title.
    11. Click the button Submit. It may take an hour or more to complete this calculation.

    12. If you are selecting sequences manually, you will see SELECT SEQUENCES.
      For instructions, see Limiting ConSurf Analysis to Proteins of a Single Function.
      Example: Sequences ready for selection (2vaa, Swiss-Prot).

    13. When your job is finished, under Final Results, click the link View ConSurf Results with FirstGlance in Jmol.
    14. Completed Example: 4enl result in ConSurf.
    15. 3 important things to take from your completed result:

      1. Explore your result in FirstGlance in Jmol. Pay attention to expected vs. unexpected conservation. Unexpected conservation can lead to new insights.

      2. Note the "APD" (Average Pairwise Distance), a measure of the diversity in the "MSA" (Multiple Sequence Alignment).
        • When APD is lower than 0.5, conservation may be exaggerated. Then, it may be worthwhile to re-run the job with more sequences. (In step (g) above, it may help to use a larger sequence database, such as UniProt or NR.)
        • When APD is higher than 1.2, the MSA may include proteins of different functions, which will obscure the conservation you are trying to see. Then, it may be worthwhile to re-run the job with fewer sequences. (In step (g) above, it may help to use a smaller sequence database, such as Clean-UniProt or Swiss-Prot.)

      3. To save your results, use Download all ConSurf outputs in a click!. You can't exactly replicate these results in future because of the constant growth of the sequence databases and upgrades to ConSurf itself. And you can upload the saved ConSurf-modified PDB file to review the results in FirstGlance.

  32. Background: see Introduction to Evolutionary Conservation.

  33. Effect of mutation on protein function Genetic consequence Example
    Function LOST** CONSERVED:
    mutation LOST from gene pool
    R133C*
    None NOT conserved:
    mutation remains in gene pool
    E143?*
    * in methyl CpG binding protein 2 (MeCP2), 3c2i:

       ASASPKQRRS IIRDRGPMYD DPTLPEGWTR KLKQRKSGRS AGKYDVYLIN
       PQGKAFRSKV ELIMYFEKVG DTSLDPNDFD FTVTGRGSPS RHHHHHH
             ^          ^

    ** R133C causes Rett syndrome, a severe neurological disorder.
    Gray: disordered in crystal, absent in model 3c2i.


    1. Enzyme example: ConSurf-colored sequence -- 4enl ConSurf Result -- enolase in Wikipedia.
    2. Multiple sequence alignments reveal conservation: MSA for 4ENL in black and white (printed handout).
    3. Detail of MSA. ConSurf does a much more sophisticated job of calculating evolutionary conservation scores than this simple example!
    4. On the less conserved side of the molecule, touch the isolated highly conserved residues to display the amino acid and sequence number. Gly236 and Pro290 are highly conserved. Why?.

  34. ConSurf's Mechanism: Simplified;   Details;   Technical.
  35. Note the Caveats in Proteopedia's Evolutionary Conservation.

  36. FirstGlance in Jmol: Easy Visualization of Any Macromolecule

    Terminology: "visualization" vs. "modeling". (Light modeling tools)

  37. To start FirstGlance, google "firstglance" (no space), or go to FirstGlance.Jmol.Org. Then enter the PDB code, or upload your homology model.

    Unusually large models may take a long time to display and be sluggish to manipulate in FirstGlance. For such cases, using Java will enable much better performance in FirstGlance. Java is not needed for most models. Ask the instructor for advice. Here are instructions for Installing and Enabling Java.

  38. Explore 1izh in FirstGlance.
    1. Introduction
    2. Molecule Information Tab
      1. Year, Method.
      2. Resolution.
      3. Free R. (1awq)
      4. Chain details.
      5. Sequences: Crystallized vs. Full Length. Alignment at UniProt (1d66).
      6. Abstract.
      7. Citations.
      8. Text contents of the PDB file.
    3. Views tab
      1. Top 3 rows of views:
        Secondary Structure / Cartoon / N->C Rainbow
        Composition / Hydrophobic/Polar / Charge..
        Local Uncertainty / Vines / Thin Backbone
      2. Buttons.
        Ligands+ / Water / Slab
      3. 1pgb: Hydrophobic core: Hydrophobic/Polar, then Slab.
      4. 1pgb: Amphipathic helices and strands. (In FirstGlance, use Isolate.. on each end of a helix or strand.)
          Potassium channel (1R3J) showing membrane surface planes (from OPM). See the Resources tab in FirstGlance.
      5. Compare with the Hydrophobic/Polar View of 1bl8 or 7ahl.
    4. Resources tab
      1. See lipid bilayer boundaries (1bl8 or 7ahl).
    5. 1pgb: Tools tab with Views.
      1. Salt bridges.
      2. Cation-pi interactions.
      3. Distances.
      4. Salt bridges in Charge View (Red sidechain (-) touching blue sidechain (+)).
      5. Charges with Slab on.
      6. Sidechain distributions in Vines View (rings buried; charges on surface).
      7. Find (review Chart of AA): PHE, (VAL,LEU,ILE), ASN, THR

  39. Explore 9ins in FirstGlance.
    1. Tools tab
      1. Disulfides/S/Se


    Contacts: Anti-Alzheimer's drug analog (*) interaction with acetylcholinesterase (Dvir et al./Sussman, 2002).
  40. Explore 3onz in FirstGlance. (Letter O not numeral zero!)
    1. Molecule Information Tab
      1. Two chains, not sequence identical.
      2. Missing residues.
      3. Ligands+ and non-standard residues
    2. Views tab
      1. Ligands button; smaller ligands.
      2. Hide (chain, toluene, isolated His).
    3. Tools tab
      1. Contacts
      2. Non-covalent interactions for HEM in chain A (blue chain).
    4. Resources tab
      1. Biological unit.

  41. Solution Nuclear Magnetic Resonance (NMR)

    Introduction to Structural Bioinformatics and Genomics
    Educators: You are welcome to use the slides linked below, or to adapt content from them into your own slides.

  42. Slides Covering:     (from bioinformatics.molviz.org)

We won't have time to go through the following resources in detail, so the links and information below are provided mostly for you to use, if you wish, after the workshop.
    Polyview-3D for Animated Powerpoint Slides and Publication-Quality Images:
      PowerPoint-Ready Animation from Polyview-3D. Click on the image for a larger view and explanation.

  1. Make Animated PowerPoint Slides and Publication-Quality Images easily with Polyview-3D.
    Proteopedia.Org (Part II): Authoring

  2. Add Molecular Scenes and Content to Proteopedia.Org
  3. Its a wiki: you or your students can add pages or customized molecular scenes in Jmol.
  4. Great for journal supplementary materials or research group websites as well as molecular structure tutorials and student reports.
  5. Protect your pages from being changed by anyone else, e.g. Nucleosomes (protected).
  6. An easy Scene Authoring Tool attaches your customized views to Green links.
  7. This is, by far, the easiest place to create molecular tutorials, e.g. Nucleosomes (publically editable).
  8. Sandboxes (enough for your entire class!) allow students to try authoring temporary molecular scenes -- without individual accounts. Screenshots can document student work. See Teaching Strategies Using Proteopedia.

  9. Jmol in Scientific Journals:

  10. Interactive 3D Complements in Proteopedia: "Supplementary Materials"

  11. FirstGlance in Jmol: Look for the buttons in Nature and Nature Structural and Molecular Biology.

  12. "Jmolized" Interactive 3D Journal Figures:
    Educational Resources

  13. Tutorials disponible en español at MolviZ.Org (Estructura del ADN; Modelo de bicapa lipídica y canal de gramicidina; Estructura del agua: enlaces de hidrógeno) and BioModel (Estructura de proteínas; Glúcidos, Lípidos, Vitaminas, Aminoácidos, Hélice alfa, Hebra beta, Lisozima, Nucleósidos, ADN, ARN, et al.)

  14. HighSchool.MolviZ.Org: Resources for High School Teachers.
  15. Toobers in Science Education!
  16. Molecular Workbench (from Concord Consortium): Activities for High School Students with built-in assessment and reporting.

    If this image is not moving, reload the page!
    Morph of the lactose repressor bending DNA as it recognizes the operon. More..
  17. Teaching Scenes, Tutorials, and Educators' Pages in Proteopedia, including Molecule of the Month.
  18. Teaching Strategies Using Proteopedia.
  19. MolviZ.Org: Martz Central: Resources for High School, College, and Researchers.
  20. Animations / Morphs: Conformational Changes (see MOVIE at right).

    Molecular Modeling (Time Permitting, or Later Private Sessions)

  21. 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.

  22. Structural Alignment of two or more chains or molecules, and how to view the alignment.


Keep in touch!
Endnotes

Endnote 1 (): Slabbed image from Electron Density at 1.0 σ showing Leu5 and Ala4 in 3hyd. Temperature factors in those two residues range from 3 to 12, average 5.

Endnote 2 (): Slabbed image from Electron Density Maps at 1.0 σ showing Leu425 and Ala426 in 1io1. Temperature factors in those two residues range from 12 to 17, average 15.

Endnote 3 (): Slabbed image with electron density map at 1.0 σ showing Ile98 and Val99 in 4cgf. Temperature factors in those two residues range from 32 to 45, average 37.