1. EGEE-II INFSO-RI-031688 Enabling Grids for E-sciencE www.eu-egee.org EGEE and gLite are registered trademarks “High throughput” protein structure prediction application in EUChinaGRID G. Minervini, G. La Rocca, P.L. Luisi and F. Polticelli Dept. of Biology, Univ. Roma Tre, and INFN Catania, Italy EGEE User Forum – Manchester, 10 May 2007

2. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 2 EUChinaGRID Project Overview EUChinaGRID project promotes the integration and interoperability between Europe (EGEE) and China (CNGrid) Grid infrastructures The goals of the EUChinaGRID are: Promoting porting of new applications on the Grid infrastructures Training of new user communities Supporting the adoption of grid tools for scientific applications Validating the intercontinental Grid infrastructure

3. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 3 Biological Applications  The protein folding “problem” and the structural genomics challenge –The combination of the 20 natural amino acids in a protein specific sequence dictates the three-dimensional structure of the entire protein –Protein function is linked to the specific three-dimensional arrangement of amino acids functional groups –With the advancement of molecular biology techniques a huge amount of information on protein sequences has been made available but far less information is available on structure and function of these proteins –The “ab-initio” prediction of protein structure is a key instrument to better understand the protein folding principles and successfully exploit the information provided by the “genomic revolution ”

4. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 4 There may be an entire universe of “Never Born Proteins” (NBP), whose properties have never been sampled by Nature Contingency theory: Extant proteins are the result of the simultaneous interplay of several concomitant causes (Gould, 1994). Determinist theory: The life constituents are the result of an evolutive fine work; what we see is the better possible solution for the biological needs (de Duve, 1995). Natural proteins Possible-protein space THEORETICAL CONTEXT

5. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 5 SOME BASIC CALCULATIONS WITH A 70 AMINO ACID PEPTIDE....  1 X 10 70 POSSIBLE AMINO ACID COMBINATIONS  SYNTHESIS OF ONE MOLECULE OF EACH COMPOUND: –1.1 X 10 42 Kg OF MATERIAL –1.8 X 10 17 TIMES THE WEIGHT OF THE EARTH  With 20 different comonomers a protein chain of just 70 amino acids can theoretically exist in 20 70 chemically and structurally unique combinations

6. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 6 The “Never Born Proteins”  It seems unlikely that nature tried all possible combinations, in other words, there exist a big number of NBP that have never been exploited by biological systems.  The NBP pose a series of interesting questions for the biology and basic science in general: –Which are the criteria with which the existing proteins have been selected? –Natural proteins have peculiar properties? (i.e. of thermal stability, solubility in water or amino acid composition?) –Or else they represent just a subset of the possible protein sequences generated only by the contemporary action of contingency and physico- chemical forces? Razionale

7. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 7 The approach  The problem is tackled by a “high throughput” approach made feasible by the use of the GRID infrastructure.  A library of 10 7 -10 9 random amino acid sequences of fixed length is generated (n=70).  “ab initio” protein structure prediction software is used  Analysis of the structural characteristics of the resulting proteins in terms of: –Frequency of compact folds and characteristics of the corresponding amino acid sequences –Occurrence of novel yet unknown folds –Hydrophobicity/Hydrophilicity characteristics –Presence of putative catalytic sites –Experimental validation on “interesting” cases

8. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 8 Never born proteins  Software –Rosetta  Developed by David Baker – University of Washington  Based on a “fragment assembly” strategy  semi-empirical force field for the evaluation of the thermodinamics of the predicted structure  Particularly successful in the prediction of novel folds in the CASP competitions (Critical Assesment of Structure Prediction) (http://depts.washington.edu/bakerpg/)

9. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 9 Rosetta Software “ab-initio” –Based on the assumption that local interactions bias the conformation of sequence fragments while global interactions select the three-dimensional structure with minimal energy, compatible with the local biases. –To define the local sequence-structure relationships the software uses the Protein Data Bank (www.rcsb.org) to extracts the most likely distribution of conformations adopted by short protein segments in experimental structures –Taking them as an approximation of the distribution adopted by sequence segments during the folding process.

10. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 10 Method details  Module I - Input generation –The query sequence is divided in fragments of 3 and 9 amino acids –The software extracts from the data base of protein structures the distribution of three- dimensional structures adopted by these fragments based on their specific sequence –For each query sequence is derived a fragments data base which contains all the possible local structures adopted by each fragment of the entire sequence.  Module II - Ab initio protein structure prediction –The sets of fragments are assembled in a high number of different conbinations by a Monte Carlo procedure. –The resulting structures are subjected to a energy minimization procedure –The principal non-local interactions considered are hydrophobic interactions, electrostatic interactions, main chain hydrogen bonds and excluded volume. –The structures compatible both with local biases and non-local interactions are ranked according to their total energy resulting from the minimization procedure.

11. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 11 Module I –The procedure for input generation is rather complex but computationally inexpensive (10 min of CPU time on a Pentium IV 3,2 GHz) –Due to the many dependencies of module I (Blast and psipred), the input generation is carried out locally with a script that automatizes the procedure for a large dataset of sequences –Approximately 500 input datasets are currently being generated weekly

12. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 12 Module II –Input –fragment files generated by module I –secondary structure prediction using psipred –In output the user obtains a number of structural models of the query sequence ranked by total energy –A single run with just the lowest energy structure as output takes approx. 10-40 min of CPU time depending on the degree of refinement of the structure

13. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 13 Integration on the GILDA facility  Single job execution on GILDA –A single Rosetta abinitio run consists of two different phases. In the first phase an initial model of the protein structure is generated using the fragment libraries. The initial model is then used as input for the second phase in which the model is refined. –A shell script has been prepared which: registers the program executable and the required input files (fragment libraries and secondary structure prediction file) on the LFC catalog calls the Rosetta executable and proceeds with workflow execution. –A JDL file was created to run the application on the GILDA working nodes which use the gLite middleware

14. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 14 Integration on the GENIUS web portal  To make easy the use of the Rosetta abinitio on Grid environment by the computational biology community, the application was integrated within the GENIUS portal (https://glite-tutor.ct.infn.it).https://glite-tutor.ct.infn.it  After a simple MyProxy server initialization procedure, input files and executable uploading, JDL file preparation, application running, run status monitoring and download of the output file are carried out from GENIUS web portal  The Rosetta abinitio output (universal.pdb format) can easily downloaded on local machine just typing download on Genius web interface. For an istant on- line check, is also provides a high resolution graphical output of the predicted structure in.png and.wrl formats is also provided

15. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 15  Step 1. After MyProxy initialization the user connects to the GENIUS portal to set up the parametric JDL, specifying the number of runs (equivalent to the number of amino acid sequences to be simulated) to be carried out.

16. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 16  Step 2. The user specifies the working directory and the name of the shell script.

17. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 17  Step 3. Input files (fragment libraries) are loaded as a single.tar.gz folder per amino acid sequence.

18. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 18  Step 4. Output files (initial and refined model coordinates) are specified in parametric form.

19. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 19  Step 5. The parametric JDL file is generated and visualized to be inspected by the user.

20. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 20  Step 6. The parametric job is submitted and its status as well as the status of individual runs of the same job can be checked.

21. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 21  Graphical output of the predicted structure representation generated in.png format by Molscript and Raster3D.

22. Enabling Grids for E-sciencE EGEE-II INFSO-RI-031688 G. Minervini EGEE User Forum * Manchester, 10-5-2007 22 CONCLUSIONS  We are currently accumulating data on NBP structures  Collecting tools for analysis (structure and function analysis)  Studying portability of other applications (e.g. function recognition software developed “in house”) in GRID  Envisioning application of ported tools for structural genomics initiatives on biomedically relevant targets –Example: prediction of the structure/function of the entire set of proteins of selected viral and microbial pathogens for target selection and in silico drug discovery

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