Two parts to successful model building

1
Two parts to successful model building

• BUILDING TOOLS knowledge and use
• Place helix here to start the path of the protein
  chain.
• Use mutateautofit to test sidechain assignments
  with
• Low energy rotamers
• standard bond lengths and angles.
• Use real space refine to
• adjust fit to e- density
• to break up steric clashes
• Planar peptide bonds.
• Validation tools to detect disallowed f and y
  angles.

• NAVIGATION SKILLS through electron density
• Recognizing structural features in electron
  density maps and skeleton maps.
• What are the distinctive features of
• .a-helix density.
• .b-strand density.
• What e- density features distinguish the
  directionality of a polypeptide?
• How to pinpoint the location of amino acid
  a-carbons.

2
Basic protein chemistry
planar peptides chiral a-carbons allowed f,y
angles- see Ramachandran plot
3
Lowest energy f,y angles correspond to a-helices
and b-sheets
b-sheet
a-helix
Ramachandran plot
Lets focus on recognizing helix and strand
features in electron density maps.
4
Helix directionality
C-terminus
Protein models are always numbered from
N-terminus to C-terminus as shown here. So it is
easy to tell the directionality of a helix.
But, electron density isnt labeled with
residue numbers. What structural features are
present in the electron density map to help you
determine which direction to place the helix?
Backbone oxygens
Backbone nitrogens
N-terminus
5
Carbonyl oxygens point to C-terminus
C-terminus
6
Cb point to the N-terminus like the branches of a
Christmas tree.
7
Cb point to the N-terminus like the branches of a
Christmas tree.
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Which way is the C-terminus pointing?
9
Test 2 Which way is the C-terminus pointing ?
10
Helices viewed from two different perspectives
Viewed down helical axis
Viewed perpendicular to helical axis
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The hole through the center of a helix is the
most distinctive feature of a-helix density.
Viewed down helical axis Often it is easier to
recognize helical density when viewed down the
helical axis due to the distinctive hole through
the center of the helix.
Viewed perpendicular to helical axis
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But, be sure to view both perspectives when
modeling an a-helix.
When viewed down the helical axis. Both
orientations of the helix appear to fit the
electron density OK.
Correct
Incorrect
13
b-strands also have directionality
From this perspective, side chains of successive
residues alternate in and out of the plane of
this page.
N-terminus
C-terminus
14
b-strands viewed from different perspectives
From this perspective, side chains of successive
residues alternate in and out of the plane of
this page.
N-terminus
C-terminus
N-terminus
C-terminus
From this perspective, side chains of successive
residues alternate up and down. Often it is
easier to recognize a beta strand by this
distinctive zig-zag pattern than by the pattern
shown above.
15
But, be sure to view both perspectives when
modeling an b-strand.
When viewed using the zig-zag perspective. Both
orientations of the strand appear to fit the
electron density OK.
incorrect
correct
But, when viewed perpendicular to the zig-zag
perspective, it becomes clear that only one
direction of the strand fits the carbonyl bumps
in the electron density.
16
b-strands always appear in sheets, b-strands are
never alone.
STRAND 1
STRAND 2
STRAND 3
STRAND 4
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Some amino acids have distinctive shapes, others
are isosteric. When in doubt, consider the
protein environment.
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Assigning the sequence
Find a stretch of 5-10 residues with well
defined side chain density. Find which amino
acid best fits the density by trial and error.
(Mutate Autofit) Keep in mind some residues
are isosteric. For example threonine and
valine. Check the proteinase K amino acid
sequence for a matching sequence of residues.
19
Proteinase K (Tritirachium album) amino acid
sequence

• 001_MAAQTNAPWG_LARISSTSPG_TSTYYYDESA_GQGSCVYVID
• 041_TGIEASHPEF_EGRAQMVKTY_YYSSRDGNGH_GTHCAGTVGS
• 081_RTYGVAKKTQ_LFGVKVLDDN_GSGQYSTIIA_GMDFVASDKN
• 121_NRNCPKGVVA_SLSLGGGYSS_SVNSAAARLQ_SSGVMVAVAA
• 161_GNNNADARNY_SPASEPSVCT_VGASDRYDRR_SSFSNYGSVL
• 201_DIFGPGTSIL_STWIGGSTRS_ISGTSMATPH_VAGLAAYLMT
• 241_LGKTTAASAC_RYIADTANKG_DLSNIPFGTV_NLLAYNNYQA

20
Rotamers
Cg
Cb
Ca
C
N
O
energetically preferred rotation angles about
single bonds in side chains
21
To control objects
Translate in x or y
Rotate around x or y
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Load map
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Calculate-gtmap skeleton
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Hide map, search for helix
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Translate helix end to center of screen (pink box)
26
Zoom and center skeleton helix
27
Rotate View (90)to look down helix axis and
re-center helix
28
Return to original view.Turn on map. Which
direction is the C-term of helix pointed?
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Calculate-gtOther Modeling ToolsPlace Helix Here.
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Saving first set of coordinates.

• File menu
• Save coordinates
• Select which coordinate set you want to save.
• 1 Helix
• Auto suggestion
• Helix-coot-0.pdb
• Change to
• sawaya-coot-0.pdb

version number
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Updated coordinates? Save with incremented
version number.

• Select save.
• 1 sawaya-coot-0.pdb
• Auto suggestion sawaya-coot-1.pdb
• Accept .
• Next time
• sawaya-coot-2.pdb,
• sawaya-coot-3.pdb, etc.

1 sawaya-coot-0.pdb
sawaya-coot-1.pdb
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Save coordinates frequently

• 1) Every 5 minutes.
• 2) When you have done some modeling that you are
  especially pleased with.
• 3) When you are fear that the next step is going
  to destroy your previous work.

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Zoom in on a distinctive side chain. Calculate
-gtModel/Fit/Refine
34
Mutate Autofit
35
Select amino acid type
36
If rotamer is incorrect, choose another
37
Sample different rotamers
Accept
38
Real Space Refinement can tidy up.
If you dont like it, Reject it. If you
accidentally accepted, then, undo.
39
Move to next residue. Mutate Auto Fit.
40
What is the next residue?
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What is the sequence of these three amino acids?
F T A Or F V A ?????
42
Proteinase K (Tritirachium album) amino acid
sequence

• 001_MAAQTNAPWG_LARISSTSPG_TSTYYYDESA_GQGSCVYVID
• 041_TGIEASHPEF_EGRAQMVKTY_YYSSRDGNGH_GTHCAGTVGS
• 081_RTYGVAKKTQ_LFGVKVLDDN_GSGQYSTIIA_GMDFVASDKN
• 121_NRNCPKGVVA_SLSLGGGYSS_SVNSAAARLQ_SSGVMVAVAA
• 161_GNNNADARNY_SPASEPSVCT_VGASDRYDRR_SSFSNYGSVL
• 201_DIFGPGTSIL_STWIGGSTRS_ISGTSMATPH_VAGLAAYLMT
• 241_LGKTTAASAC_RYIADTANKG_DLSNIPFGTV_NLLAYNNYQA

43
Extend N and C termini one amino acid at a time

• Center on the N or C-terminus of the helix
• Click on Add Terminal Residue
• Accept or drag to better location.

44
Remember

• Do not use maximize button to expand the Coot
  window to full screen mode. It will hide pop up
  dialog boxes.
• Coot will be waiting for a response, but youll
  never see the question because it is hidden
  behind the full screen window.
• Instead, stretch window by dragging corner.

NO!
drag corner
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Remember

• Recognize the location of a-carbons.
• Dont confuse sidechain and mainchain density.

46
Remember

• Recognize the location of a-carbons.
• Dont confuse sidechain with mainchain density.

47
Save coordinates frequently or suffer set backs.
48
Results
The last version number of each baton build
session represent your best effort at modeling
that segment of the protein chain. Next week, we
will concatenate these segments of chain into one
file and refine them, calculate Rwork and Rfree.

• sawaya1-coot-0.pdb
• sawaya1-coot-1.pdb
• sawaya1-coot-2.pdb
• sawaya1-coot-3.pdb
• sawaya1-coot-4.pdb
• sawaya1-coot-5.pdb
• sawaya1-coot-6.pdb
• sawaya1-coot-7.pdb
• sawaya1-coot-8.pdb