Exercise 1
Analysis of structure quality and basic structural characteristics

1. Load the structure of carbonmonoxy myoglobin (MbCO) with code 1MBN (historical example)
   Note that some side chains are re-constructed because of lack of atom coordinates - this is because of lack of electron density, or high temperature factors, or low resolution, characteristic of flexibility
   What is the resolution of this structure and how old is it?
   Hint: Click on the "text button" of Tool Bar (3d raw, 2nd button) - This options opens a text version of the PDB file
   Answer: 2 Å and deposited in 1973
2. Backbone structure quality
   Inspect the Ramachandran Plot
   In Pull Down Window select Ramachandran Plot
   In Tool Bar click on Select -- All
   Are there any residues in dissallowed regions?
   Hint: place mouse on dots in Ramachandran Plot and read the residue name and number
   Answer: yes, Gln152 - note special status of Gly resides
   In Tool Bar Select -- Group kind "Gly"
   Is this structure mainly helical, mainly beta sheet, or mixed?
   Hint: Check the Control Panel Window, 1st column for additional help
   Answer: all helical
   In Tool Bar Select -- Secondary structure "helices, strands, coils"
3. Side chain structure quality
   Find amino acid clashes
   Hint: Select -- Amino acids making clashes and look at residues colored in red in Control Panel
   Clashes between atoms are shown in magenta dashed lines
   Answer: Gln26, Ile30, Lys78, Glu85, His93, Lys145, Glu148, Hem1
   Repeat Select -- Amino acids making clashes with the backbone
   Answer: none
   Close structure
4. Load the structure of carbonmonoxy myoglobin (MbCO) with code 1A6G
5. Check quality of backbone structure by inspecting the Ramachandran Plot
   Answer: Gln152 is again in a disallowed region
6. Identify amino acid clashes
   Answer: only His93 (proximal histidine) with HEM1 (heme prosthetic group), which is expected since His93 and Hem1 are covalently linked
7. Hydrogen bonding quality
   Find side chains lacking proper hydrogen bond
   Hint: use Select
   Note that hydrogen bonds are marked with green dashed lines
   Answer: several colored in red in Control Panel
8. Quality of a helix: Helix E
   Hide structure with the exception of Helix E
   Hint: Select -- None (red color disappears from Control Panel residues)
   Hint: keep Ramachandran Window open
   click on "group" in second column of List Title of Control Panel (structure disappears in Main Window)
   Identify Helix E in Control Panel Window
   Click on 1st residue of Helix E in Control Panel Window and drag down to include (select) all residues of Helix E
   Identify side chains in helix E (fifth helix) that lack proper hydrogen bond
   Answer: Asp60, Lys62, Lys63, Thr67, Thr70
   Remove the side chains and visualize backbone hydrogen bond donors/acceptors
   You can also check H-bond distances using Display -- H-bond distances
   Remove H-bonds and H-bond distances using Display
   Check if Helix E is a good amphipathic Helix
   Hint: orient Helix E in a helical wheel-type of orientation
   Add ribbon to better visualize helix (use 6th column of Control Panel Window- drug to include all residues of Helix E)
   Identify the polar side of the Helix (check for side chains with N-atoms (blue) or O-atoms (red) in side chains)
   Identify the hydrophobic part of Helix E
   Answer: yes, helix E is a good amphipathic helix
9. Heteroatoms
   Identify the Heme prosthetic group and the carbon monoxide, remove the rest of the protein
   Add Helix E (5th) and Helix F (6th) all backbone, side chains, and ribbons
   Select Ribbon by clicking in the arrow head of the 8th column of the Control Panel
   Select the Ribbon Color by clicking or drugging the mouse on the squares corresponding to residues of Helices E and F
   Select color yellow (it looks good on black background)
   In Preferences -- Ribbons -- click on "Render as solid ribbon"
   In Display click on Use openGL rendering and Render in solid 3D
   Voila!
10. Add hydrogens! Proteins have hydrogen, crystallographic structures don't (in most cases)!
    Build -- Add hydrogens
11. Calculate distances, angles, torsion angles
    Make sure that residues of Helices E and F are selected in Control Panel
    Identify His64
    Hint: use button "Leu?" (8th from left) of Toolbar
    Hint: to get rid of labels, use Display -- Label type -- "ClearUserLabels"
    There are 2 His residues present, click on one, is it His64?
    Measure the distance between the O-atom of CO and the NH of the ring of His64
    Hint: use 5th button of Toolbar with the icon of a distance
    Answer: 3.16 Å
    Measure the angle of Fe-C-O
    Hint: use the 6th button of Toolbar - you need 3 atoms to define an angle - click on the central atom first and follow with the other two
    Answer: 171,15 degrees
    Measure the backbone torsion angles of His64
    Hint: use the 7th button of Toolbar
    Answer: phi=-69.50, psi=-32.46 omega=-179.30
    Helpfull hint: If the "caps lock" key is pressed, you can make several successive measurements
12. Water molecules
    Add water molecules, if present in structure!!!
    Check if PDB file contains waters using the text icon on bottom left of Toolbar
    Click Build -- Add H2O
    Click the O-atom of CO
    Repeat by clicking the N-atom or HN-atom of His64 side chain
    How many water molecules are in the heme pocket between CO and His64 (distal His)?
    Answer: 2
    You can alsa modify Preferences -- Loading protein to show water molecules upon loading the protein
13. Solvent salt molecules
    How many solvent sulfate molecules are present
    Answer: 2
14. C-terminal O-atom
    Typically present as a separate group at end of sequence
15. Go back to full structure
    Select -- None , and get rid of all selected residues by clicking on 2nd column of Control Panel
    Select -- All , and bring back the structure by clicking on 2nd column of Control Panel
    Unselect all residues again
    Center molecule by clicking on 1st button of Tool Bar
    Get rid of ribbon
    Get rid of H-bonds in Display -- Show H-bonds