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MODELLER WORKSHOP ITERATIVE EXAMPLE

The alignment-modeling-evaluation cycle. The case of the Holoferax volcanii dihydrofolate reductase. (needed files)

Several structures of dihydrofolate reductase (DHFR) are known. However, the structure of DHFR from Haloferax volcanii was not known and its sequence identity with DHFRs of known structure is rather low ~30%. A model of H. volcanii DHFR (HVDFR) was constructed before the experimental structure was solved. This example illustrates the power of the iterative alignment-modeling-evaluation approach to comparative modeling.

Of all the available DHFR structures, HVDHFR has the sequence most similar to DHFR from E. coli. The PDB entry 4DFR corresponds to a high resolution (1.7Å) E. coli DHFR structure. It contains two copies of the molecule, named chain A and chain B. According to the authors, the structure for chain B is of better quality than that of chain A. The following TOP file aligns HVDFR and chain B of 4DFR.

READ_MODEL FILE = '4dfr.pdb', MODEL_SEGMENT '@:B' 'X:B'
SEQUENCE_TO_ALI ALIGN_CODES = '4dfr'
READ_ALIGNMENT FILE = 'hvdfr.seq', ALIGN_CODES = 'hvdfr', ADD_SEQUENCE = on
ALIGN2D
WRITE_ALIGNMENT FILE='hvdfr-4dfr.ali'
WRITE_ALIGNMENT FILE='hvdfr-4dfr.pap', ALIGNMENT_FORMAT = 'PAP', ;
ALIGNMENT_FEATURES = 'indices helix beta'

File: align2d-4.top

Some options used in this example include MODEL_SEGMENT, which is used to indicate chain B of 4DFR; and ALIGNMENT_FEATURES, which is used to output information such as secondary structure, to the alignment file in the PAP format.

_aln.pos 10 20 30 40 50 60
4dfr M-ISLIAALAVDRVIGMENAMPW-NLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRK
hvdfr MELVSVAALAENRVIGRDGELPWPSIPADKKQYRSRIADDPVVLGRTTFESMRDDLPGSA
_helix 999999999999 999999999
_beta 9 999999999 999999 999

_aln.pos 70 80 90 100 110 120
4dfr NIILSSQPGT--DDRVTWVKSVDEA--IAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTH
hvdfr QIVMSRSERSFSVDTAHRAASVEEAVDIAASLDAETAYVIGGAAIYALFQPHLDRMVLSR
_helix 99999 99999 99999999
_beta 99999 99999 9999999 9999999

_aln.pos 130 140 150 160
4dfr IDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFKILERR
hvdfr VPGEYEGDTYYPEWDAAEWELDAETDHEGF---TLQEWVRSASSR
_helix
_beta 99 999999999999 999999999999

File: hvdfr-4drf.pap

Using the PIR alignment file hvdfr-4dfr.ali, an initial model is calculated.

INCLUDE
SET ALNFILE = 'hvdfr-4dfr.ali'
SET KNOWNS = '4dfr'
SET SEQUENCE = 'hvdfr'
SET STARTING_MODEL = 1
SET ENDING_MODEL = 1
CALL ROUTINE = 'model'

File: model4.top

Because the sequence identity between 4DFR and HVDFR is relatively low (30%), the automated alignment is likely to contain errors. The PROSA evaluation of the model shows two regions with positive energy.

PROSAII profile for model initial model

The first region is around residue 85, the second region is at the C-terminal end of the protein. Referring to the target--template alignment above (hvdfr-4dfr.pap), it is easy to understand why the first positive peak appears. The insertion between position 85 and 88 of the alignment was placed in the middle of an α-helix in the template (the "9" characters on the first line below the sequence mark the helices). Moving the insertion to the end of the α-helix may improve the model. The second problem, which occurs in the C-terminal region of the alignment, is less clear. The deletion in that region of the alignment corresponds to the loop between the last two β-strands of 4DFR (a β-hairpin). Since the profile suggests that this region is in error, an alternative alignment should be tried. One possibility is that the deletion is actually longer, making the C-terminal β-hairpin shorter in HVDFR. One plausible alignment based on this considerations is shown here.

_aln.pos 10 20 30 40 50 60
4dfr M-ISLIAALAVDRVIGMENAMPW-NLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRK
hvdfr MELVSVAALAENRVIGRDGELPWPSIPADKKQYRSRIADDPVVLGRTTFESMRDDLPGSA
_helix 999999999999 999999999
_beta 9 999999999 999999 999
ß
_aln.pos 70 80 90 100 110 120
4dfr NIILSSQPGT--DDRVTWVKSVDEAIAACG--DVPEIMVIGGGRVYEQFLPKAQKLYLTH
hvdfr QIVMSRSERSFSVDTAHRAASVEEAVDIAASLDAETAYVIGGAAIYALFQPHLDRMVLSR
_helix 9999999999 99999999
_beta 99999 99999 9999999 9999999

_aln.pos 130 140 150 160
4dfr IDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFKILERR----
hvdfr VPGEYEGDTYYPEWDAAEWELDAETDHE-------GFTLQEWVRSASSR
_helix
_beta 99 999999999999 999999999999

File: hvdfr-4dr-2.pap

A new model was calculated. Its PROSA profile is shown in the next figure.

PROSAII profile for model final model

Both positive peaks disappeared and the new profile does not contain any positive regions. Next figure shows the comparison of the C-terminal beta-hairpin of both models and the actual experimental structure. This confirms that the correct choice for the final alignment was made and that PROSA was indeed able to detect the error in the initial alignment.

 

Last modified: June 2004