Two parts to successful model building

• BUILDING TOOLS –how to use Coot– Initiate trace of protein chain

(“Place helix here”)– Test sidechain assignments.

• Mutate+autofit• Select rotamers

– Maintain proper geometry• “Regularize” bond lengths

and angles, planes, eliminate steric clash.

• “Real space refine” to adjust fit to e- density

– Validation tools to detect disallowed and angles.

• PATTERN RECOGNITION SKILLS– Recognizing structural

features in electron density maps and skeleton maps.

• .-helix density.• .-strand density. • Recognize -carbons

positions• Recognize side chain

density

Basic protein chemistry: Phi() and Psi()

planar peptideschiral -carbonsallowed angles-

see Ramachandran plot

CC

OC

O

N

Ni+1

Oi-1carbonnitrogenoxygen

an amino acid residue

Lowest energy angles correspond to -helices and -sheets

-helix

-sheet

Lets focus on recognizing helix and strand features in electron density maps.

Ramachandran plot

Be able to recognize a helix from different perspectives

Viewed down helical axis

Viewed perpendicular to helical axis

90o

Viewed perpendicular to

helical axis

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.

The hole through the center of a helix is the most distinctive feature of -helix density.

But, be sure to view both perspectives when modeling an -helix.

When viewed down the helical axis. Both orientations of the helix appear to fit the electron density OK.

But, when viewedperpendicular to thehelical axis, it becomes clear that only one direction of the helix fits the carbonyl bumps in theelectron density.

Incorrect Correct

Helix directionality

1

2

3

45

6

78

910

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 isn’t labeled with residue numbers.

What structural features are present in the electron density map to help you determine which direction to place the helix?

C-terminus

N-terminus

Backbonenitrogens

Backboneoxygens

Carbonyl oxygens point to C-terminus

1

2

3

45

6

78

910

C-terminus

Look forcarbonylbumps

Cpoint to the N-terminus like the branches of a Christmas tree.

C

CC

CCC

CC

CC

C pointto N-terminus

C C

C C

C C

C C

C C

Cpoint to the N-terminus like the branches of a Christmas tree.

C C

C C

C C

C C

C C

Which way is the C-terminus pointing?

Test 2: Which way is the C-terminus pointing ?

-strands appear as parallel tubes of density spaced 4.8 Å apart.

-strands are never alone.

STRAND 1

STRAND 2

STRAND 3

STRAND 4

4.8 Å

4.8 Å

4.8 Å

-strands also have directionality

N-terminus C-terminus1 3 5 7 9

2 4 6 8 10

From this perspective, side chains of successive residues alternate in and out of the plane of this page.

-strands viewed from different perspectives

N-terminus C-terminus1 3 5 7 9

2 4 6 8 10

N-terminus C-terminus

From this perspective, side chains of successive residues alternate in and out of the plane of this page.

90o

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.

2 4 6 8 10

1 3 5 7 9

But, be sure to view both perspectives when modeling a -strand.

When viewed using the zig-zag perspective. Both orientations of the strand appear to fit the electron density OK.

correct incorrect

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.

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

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.Phe - Thr - Ala- Ser

or or Val Cys

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

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

Some amino acids have distinctive shapes, others are isosteric. When in doubt, consider the protein environment.

Load Coordinates• File -> Open Coordinates--browse window

opens – Click “filter” (will show only .pdb files)– Click “sort by date” (will place the lastest

coordinates at the top of browser)– Select the .pdb file (e.g. sawaya-coot-0.pdb)

Open CoordinatesAuto Open MTZOpen MTZ, mmcif, fcf, or phs

• File -> Open mtz, mmcif, fcf, or phs– Click “filter” (will show only .mtz or .hkl

or .phs files)– Click “sort by date” (will place the

lastest .phs file at the top of browser)– Select the .phs file siras-pcmbs.phs

Open CoordinatesAuto Open MTZOpen MTZ, mmcif, fcf, or phs

Load Structure Factors (map)

siras-pcmbs.phs

Zoom in on a distinctive side chain. Calculate ->Model/Fit/Refine

Mutate & Autofit

Select amino acid type

If rotamer is incorrect, choose another

C H

H

HH

Henergetically preferred rotation angles about single bonds in side chains

CC

C

N

C

O

Rotamers

C

N

O=CH

H

H

Nearly eclipsed. Unfavored.

C

NO=C

H

H

H

A rotamer is energetically favorable because it is one of 3 possible staggered conformations.Newman Projections

NO=C

H

C

H HH

H

H

H

NO=C

H

Sample different rotamers

Accept

Real Space Refinement can tidy up.

If you don’t like it, Reject it. If you accidentally accepted, then, “undo”.

If molecule explodes, adjust refinement weight.

Lower numbers tighten geometric restraints

5.0

Move to next residue. Mutate & Auto Fit.

What is the next residue?

What is the sequence of these three amino acids?

F T A

Or

F V A

?????

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

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.

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 you’ll never see the

question because it is hidden behind the full screen window.– Instead, stretch window by dragging corner.

X

NO!

X

drag corner

X

Save coordinates frequently or suffer set backs.

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

1

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

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.

Results

• 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

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.

To control objects

Rotate around x or y

Translatein x or y