The document is a modelling assignment focused on predicting the 3D structure of a protein from its amino acid sequence using various computational methods, namely threading and ab initio modeling. It details the use of tools like Phyre2 and Robetta for structure prediction, alignment, and validation of the generated model, culminating in the identification of the protein as a multidrug-resistance transporter. Validation was performed using various servers, including Anolea and ProSA, with results indicating a successful model of the chosen amino acid sequence.

1. Modelling Assignment
Submitted to: Submitted by:
Dr. Durg Vijay Singh Shweta Kumari
Roll- 21
M.Sc. Bioinformatics
2Nd
sem

2. CONTENT

Objective

Structure prediction

Threading

Ab inito

Phyre2 and result

Dali str-str alignment and its result

Robetta and its result

Validation

Result and discussion

Conclusion

reference

3. OBJECTIVE
To build the model of given amino acid residue sequence and validate
the generated model.
>gi|407259499|gb|AFT91383.1| EcdL [Emericella rugulosa]
MDDSPWPQCDIRVQDTFGPQVSGCYEDFDFTLLFEESILYLPPLLIAASVALLRIWQL
RSTENLLKRSGLLSILKPTSTTRLSNAAIAIGFVASPIFAWLSFWEHARSLRPSTI
LNVYLLGTIPMDAARARTLFRMPGNSAIASIFATIVVCKVVLLVVEAMEKQRLLLD
RGWAPEETAGILNRSFLWWFNPLLLSGYKQALTVDKLLAVDEDIGVEKSKDEIRRR
WAQAVKQNASSLQDVLLAVYRTELWGGFLPRLCLIGVNYAQPFLVNRVVTFLGQPD
TSTSRGVASGLIAAYAIVYMGIAVATAAFHHRSYRMVMMVRGGLILLIYDHTLTLN
ALSPSKNDSYTLITADIERIVSGLRSLHETWASLIEIALSLWLLETKIRVSAVAAA
MVVLVCLLVSGALSGLLGVHQNLWLEAMQKRLNATLATIGSIKGIKATGRTNTLYE
TILQLRRTEIQKSLKFRELLVALVTLSYLSTTMAPTFAFGTYSILAKIRNMTPLLA
APAFSSLTIMTLLGQAVSGFVESLMGLRQAMASLERIRQYLVGKEAPEPSPNKPGV
ASTEGLVAWSASLDEPGLDPRVEMRRMSSLQHRFYNLGELQD

4. Structure Prediction
Protein structure prediction is the prediction of the three-
dimensional structure of a protein from its amino acid
sequence i.e, the prediction of its folding and its
secondary, tertiary, and quaternary structure from its
primary structure.
The knowledge of the 3D structure is useful for rational
drug design, protein engineering, detailed study of
protein –bio-molecular interactions, study of
evolutionary relationship between proteins or protein
families etc.

5. METHOD OF STRUCTURE PREDICTION
Structure prediction
Experimental Method Computational Method
X-Ray NMR EM Template based Template free
Homology Threading Ab inito

6. 
We have to build the model of given sequence, 604 AA
residue of Ecdl (Emerucella rugulosa).

Hence, the given protein sequence have not shown the
significant alignment with any solved structure
We cann't perform Homology Modelling to
build the given sequence.

The only alternative way is THREADING or AB
INITIO method.

8. Threading
“Remote Homology”

Method of protein modeling which is used to model those proteins
which have the same fold as proteins of known structures, but do not
have homologous proteins with known structure.

The software used for fold recognition methods are:
 PHYRE2
 I-TASSER
 MUSTER
 RaptorX
 GenThreader
 LOMETS

9. Ab inito method
Predicting the 3D structure without any “prior knowledge”
If structure homologues (occasionally analogues) do not exist, or
exist but cannot be identified, models have to be constructed from
scratch. This procedure, called ab initio modelling.

Software used for Ab inito structure prediction
 Robetta

10. PHYRE2( Protein Homology/analogY
Recognition Engine V 2.0)

Developed by Dr. Kelly

Released on 14th feb 2011.

Most popular structure prediction server cited over 1500
times.

Ranked as best for function prediction in CASPs 9.

The basic principal of work of PHYRE2 is
 Finding a sequence alignment to a known
structure.
 Copying the co-ordinate and relabeling the
residues according to our sequence based on
alignment.

11. PHYRE2

Features of PHYRE2:
 Domain analysis
 Highlight motif
 Transmembrane helix are coloured

Algorithm used to predict 3D str is LOCAL ALIGNMENT
&HMM.

Localy aligned our seq against fold library and HMM matching
of our seq and known sequence structure.

Return a confident prediction for a subsequence of our seq cut
this all confident seq and resubunit to join them for their
assembly.

12. PHYRE2 result

13. PHYRE2 best model

14. PHYRE2

15. ALIGNMENT OF QUERY TO 4f4cA

16. PHYRE2 BEST MODEL

17. TRANSMEMBRENE REGION
ANALYSED BY PHYRE2

18. DALI(Distance mAtrix aLIgnment)

Method for structure-structure alignment.

It uses 3D cartesian coordinate of c-alpha carbon atom of each
protein in order to calculate residue-residue diatance matrix.

Output generate:
 Rank of PDB identifier
 Z-score
 RMSD
 Lali (number of aligned position)
 Nres (number of aligned residue)
 %ID
 PDB discription

19. DALI result

20. DALI result analysis

Low rmsd and high nres shows the better alignment.

If both rmsd and nres is high or low, not possible to establish an
order between the alignment.

Rmsd- It is the measure of the average deviation in distance between
aligned alpha carbons (i.e, calculate the divergance from one to
another b/w two sequences)

Z score- The Z-Score is the measure of quality of the structural
alignment.
Note:- DALI package is based on Fartran programming and perl script.
“The shows the best alignment with 4f4c_A with low rmsd 0.6 and
high lali score 403.”

21. STRUCTURE-STRUCTURE
ALIGNMENT BY PYMOL

22. Ab inito through ROBETTA

Non query templete based alignment

Robetta secure the best position in CASP (Critical Assessment of
Techniques for Protein Structure Prediction) 4, 5, 6, 7 and 8.

Roberta prediction type-
 1. Ginzu : Domain prediction
 2. Structure : 3D Model (available per domain after
Ginzu completes from result page)

23. Domain prediction by GINZU protocal

There are several model Robetta produces.

It determine more than one domain that means Robetta breaks up the
query sequence into putative domains and model each of them
separately.

After that assembles all the model into contiguous chain.

24. RESULT OF ROBETTA

25. Robetta result analysis

Robetta shows the alignment with these three protein for domain
prediction:
Sl. no. Protein ID Discription
1 4p79 Crystal str of cloudin provides insight into the architecture
of tight junction
Ion channel regulator, alpha helical
Membrane protein
2 1ni0 Hydrolase
Restriction endonuclease PuvII from proteus vulgaris,
class alpha/beta protein
EC 3.1.21.4
3 4m1m Multidrug resistant protein
ATP binding cassate transpoter
Pgp

26. VALIDATION of MODEL

ANOLEA

PROSA

PROCHECK(PDBsum)

27. ANOLEA

Atomic Non-LOcal Environment Assessment

Perform energy calculation on a protein chain evaluating non-local
environment of each heavy atomin the molecule.

Steps-

1. Open anolea server

2. Browse sequence file

3. Fill job title n submit to servet .

28. ANOLEA result

29. PROSA

PROtein Structure Analysis

Developed by Sippl,1993.

Calculate quality score of C alpha carbon of input structure.

OUTPUT-
 Z score
 Plot of residue score-
 3D structure of input protein

30. PROSA

PROtein Structure Analysis

Developed by Sippl,1993.

Calculate quality score of C alpha carbon of input structure.

OUTPUT-
 Z score
 Plot of residue score-
 3D structure of input protein

31. PROSA
1 .Z score- indicate the overall quality of model value display of
all experimentally determined protein chain in PDB.
“more negative value more accurate structure”.
2. Plot of residue score- shows local quality of model by plotting
energy as sum of AA sequence position i (take window size 40)
Positive value correspond problematic or erroneous part of
structure.
3. Prosa web visualized the 3D structure of input protein using the
molecular viewer Jmol.
Residue are colored from blue to red in order of increasing residue
energy.

32. PROSA RESULT

33. PROSA RESULT

34. PROCHECK(PDBsum)

The PDB sum is a pictorial database that provides an at-
a-glance overview of the contents of each 3D structure
deposited in the Protein Data Bank (PDB).

The PROCHECK analyses provide an idea of the stereo-
chemical quality of all protein chains in a given PDB
structure.

They highlight regions of the proteins which appear to
have unusual geometry and provide an overall
assessment of the structure as a whole.

PDBsum uses version 3.6.2 of PROCHECK.

35. PROCHECK(PDBsum)

The PDB sum is a pictorial database that provides an at-
a-glance overview of the contents of each 3D structure
deposited inthe Protein Data Bank (PDB).

The PROCHECK analyses provide an idea of the
stereochemical quality of all protein chains in a given
PDB structure.

They highlight regions of the proteins which appear to
have unusual geometry and provide an overall
assessment of the structure as a whole.

PDBsumuses version 3.6.2 ofPROCHECK.

36. PROCHECK

37. PROCHECK result

38. PROCHECK result

39. PROCHECK ANALYSIS
• G factor- The G-factor is a log-odds score based on the observed
distributions of these stereo-chemical parameters.
• A low G-factor indicates that the property corresponds to a low-
probability conformation.
• These are the stereo-chemical property:
1. planarity
2. chirality
3. phi/psi preferences
4. chi angles.

40. Result and discussion

Fold recognition was done through PHYRE2 server for fold
assessment.

On the other hand ab initio prediction was analyzed by Robetta
sever which gives information about domain.

After build the model, model was validated through some
server ANOLEA & PROSA.

Ramachandran plot of model analysed using PDBsum
PROCHECK with the description of the allowed region.

41. Result and discussion

The comparative and combined study of phyre2 and Robetta
shows:-
Sl. no.Sl. no. Str. Prediction methodStr. Prediction method Protein idProtein id discription
1 Fold recognition by PHYRE2 4F4C Crystal structureof
themultidrugtransporterP-
glycoprotein from C.
elegans
2 Ab initio by Robetta 4p79 Membrane protein
3 Ab initio by Robetta 1ni0 Hydrolase
4 Ab initio by Robetta 4m1m Multidrug resistant
protein,ATP binding
cassate transpoter,Pgp

42. Conclusion

The above results of PHYRE2 (fold recognition method) and Robetta
(ab initio prediction) generate the model of given AA sequence which
conclude that the given protein is
 P-glycoprotein: multidrug-resistance and a
superfamily of membrane-associated transport
proteins.
 ABC (ATP binding cassette) transporter
 Transmembrane protein (alpha helical structure)

43. References

http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index

http://www.sbg.bio.ic.ac.uk/phyre2/phyre2_output/7330b2b464c1ea64/summary.html

http://robetta.bakerlab.org/

http://melolab.org/anolea/

https://prosa.services.came.sbg.ac.at/prosa.php

http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html

http://ekhidna.biocenter.helsinki.fi/dali_server/results/20150324-0049-69ef51112579617192cac4dcad7075f2/index.html