Tuesday, August 29, 2006
Circular Dichroism Analysis
This is the Dicroweb, which a facility of the BBSRC Centre for Protein and Membrane Strucutre Dynamics, based in the UK. This site provides on-line Circular Dichroism (CD) analysis. I personally found it to be a great tool for CD analysis. Easy to up-load your raw data, with the web-site performing all the calculations for you (inputing mdeg and getting ellipticity measurements back (rme or other)). You do need to read a little about the types of analysis that this site offers as there are several different models to use, (CONTINLL, SELCON3, CDSSTR, VARSLC, and K2D) as well as different reference sets. But if you are doing Circular Dichroism, then you really should learn what these are (limitations etc). You will need an account, which in my case was granted in about a week or so (maybe less, it was pretty quick though).
My only problem with it was that I was analysing a 9 amino acid peptide and this site has a maximum time-limit for its analysis as they don't want to bog the server down too much. For normal proteins etc this is an excellent program and I give it my full support. For peptides though, you are trying your luck. Give it a go though, you may get lucky.
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CDPro is kind of a gold standard in CD analysis from Colorado State University. It is stand-alone, so you can download it and run it on your own computer (don't know if it runs on Macs not running Boot-camp though). It runs in your internet browser (Netscape or Internet Explorer) and uses the CONTINLL, SELCON3, and CDSSTR programs for analysis. It's use is free for not-for-profit organizations. I would try this as well, there is a new "membrane proteins" reference set included now which may be of use.
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PEPFIT. This program's development is detailed in:
Reed, J. & Reed, T.A. (1997) A set of constructed type spectra for the practical estimation of peptide secondary structure from circular dichroism. Anal. Biochem. 254, 36±40.
and it is my favorite. There is no link here because you need to get in contact with the authors of this program yourself and they will send it to you. It is an MS DOS based program designed by the authors to help in their own research, and was designed based on CD specs from peptides (as opposed to the large globular proteins used in other CD analysis programs). It allows you to guess what percentage of several types of conformations your spectra may have, and then the program overlays your guess with your actual result and gives you an error calculation.
While it's a great program, I didn't like the heavy requirement of user input, and it may take you a while to get a good fit if your peptide is a mixture of several types of beta turns.
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PEPFIT Analysis. This is a program written by myself and is entirely based on the PEPFIT program described above. This program is also stand alone, but has the advantage of running in MS Office (Excel). I have tested it in all versions of Office since 2000, and it works in all including Office 2007. You must allow macros to be run. It was written in Excel 2000, and runs best there though.
The advantages of the PEPFIT Analysis program over the original PEPFIT program is that it runs in Excel, and it is a point and click operation with minimal user intervention. I have kept the original idea of PEPFIT and allowed the user to guess the percent conformation, but I have also made the program do the guess-work for you. You simply paste the spectra values in, click the PEPFIT Analysis icon, select a few criteria (described in the accompanying documents), and away you go. You can analyze your circular dichroism data agianst all 9 reference curves, or select a few, but again, these details are described in the instruction supplied.
Your CD results are compared with combinations of the reference curves you select, and the final top 10 best fit results are presented using an R squared error calculation as the best fit criteria.
The first (well second if you include my thesis) is now in peer reviewed publication (Amon, M. A., Ali, M., Bender, V., Hall, K., Aguilar, M.-I., Aldrich-Wright, J., and Manolios, N. (2008) Kinetic and conformational properties of a novel T-cell antigen receptor transmembrane peptide in model membranes. Journal of Peptide Science).
Following suggestions from one user in particular, I am currently upgrading the program a bit by making it run a bit faster and simpler, and also, at the suggestion of one user, I am trying to add a reference curve fora polyproline helix, but this may take me a while as it's now something I do in my "spare" time while post-docking.
If you are interested in this software, you need only ask for a copy (post a comment or e-mail michael_amon@hotmail.com and use "PEPFIT Analysis" as your subject heading.
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My only problem with it was that I was analysing a 9 amino acid peptide and this site has a maximum time-limit for its analysis as they don't want to bog the server down too much. For normal proteins etc this is an excellent program and I give it my full support. For peptides though, you are trying your luck. Give it a go though, you may get lucky.
__________________________________________________________
CDPro is kind of a gold standard in CD analysis from Colorado State University. It is stand-alone, so you can download it and run it on your own computer (don't know if it runs on Macs not running Boot-camp though). It runs in your internet browser (Netscape or Internet Explorer) and uses the CONTINLL, SELCON3, and CDSSTR programs for analysis. It's use is free for not-for-profit organizations. I would try this as well, there is a new "membrane proteins" reference set included now which may be of use.
__________________________________________________________
PEPFIT. This program's development is detailed in:
Reed, J. & Reed, T.A. (1997) A set of constructed type spectra for the practical estimation of peptide secondary structure from circular dichroism. Anal. Biochem. 254, 36±40.
and it is my favorite. There is no link here because you need to get in contact with the authors of this program yourself and they will send it to you. It is an MS DOS based program designed by the authors to help in their own research, and was designed based on CD specs from peptides (as opposed to the large globular proteins used in other CD analysis programs). It allows you to guess what percentage of several types of conformations your spectra may have, and then the program overlays your guess with your actual result and gives you an error calculation.
While it's a great program, I didn't like the heavy requirement of user input, and it may take you a while to get a good fit if your peptide is a mixture of several types of beta turns.
_________________________________________________________
PEPFIT Analysis. This is a program written by myself and is entirely based on the PEPFIT program described above. This program is also stand alone, but has the advantage of running in MS Office (Excel). I have tested it in all versions of Office since 2000, and it works in all including Office 2007. You must allow macros to be run. It was written in Excel 2000, and runs best there though.
The advantages of the PEPFIT Analysis program over the original PEPFIT program is that it runs in Excel, and it is a point and click operation with minimal user intervention. I have kept the original idea of PEPFIT and allowed the user to guess the percent conformation, but I have also made the program do the guess-work for you. You simply paste the spectra values in, click the PEPFIT Analysis icon, select a few criteria (described in the accompanying documents), and away you go. You can analyze your circular dichroism data agianst all 9 reference curves, or select a few, but again, these details are described in the instruction supplied.
Your CD results are compared with combinations of the reference curves you select, and the final top 10 best fit results are presented using an R squared error calculation as the best fit criteria.
The first (well second if you include my thesis) is now in peer reviewed publication (Amon, M. A., Ali, M., Bender, V., Hall, K., Aguilar, M.-I., Aldrich-Wright, J., and Manolios, N. (2008) Kinetic and conformational properties of a novel T-cell antigen receptor transmembrane peptide in model membranes. Journal of Peptide Science).
Following suggestions from one user in particular, I am currently upgrading the program a bit by making it run a bit faster and simpler, and also, at the suggestion of one user, I am trying to add a reference curve fora polyproline helix, but this may take me a while as it's now something I do in my "spare" time while post-docking.
If you are interested in this software, you need only ask for a copy (post a comment or e-mail michael_amon@hotmail.com and use "PEPFIT Analysis" as your subject heading.
_________________________________________________________
Tuesday, August 22, 2006
Gene function and pathway analysis sites
This is a series of links which are helpful in analysing gene funtions by either entering a single Gene ID, or by entering a bunch of genes (gene lists from microarray analysis for eg). Just click the Blue word in each description and it will take you there.
If you find another site that you have found helpful, please let me know and I will add it to this list. Hope this helps though!
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This is a link to the SOURCE website which allows you to input your gene (as a GenBank Accession number for eg) and get the gene name, symbol, ontologies, function, and several other potentially useful information. It is possible to do a single gene search, or a batch analysis. It is also possible to download your results as a tab-delimited .txt file.
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This is a link to the DAVID Bioinformatics website, which allows you to do similar things as the SOURCE website such as functional annotation. This site has several other useful features such as producing "Enrichment" scores, which, to my understanding, is an analysis of the likely-hood of a group of genes being found together. Or conversely, it gives you a numerical interpretation as to the likely-hood that these genes would come up together in a list at random. The results that this program produces all have links as well as many other useful statistical analysis. Well worth a look. (Performs an EASE analysis)
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This is another DAVID analysis website which allows you to post in gene lists from microarray experiments (or other experiment producing lists of genes) and results in sorting them into different functional categories based on their GO (Gene Ontology) Terms. It produces Annotation charts, GO Charts, Kegg Charts, Domain Charts, and provides access to EASE online (once you submit a gene list). You can also download EASE to run on your own computer from this site by following the links. I gathered alot of useful information from this website, and recommend it.
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The Gene Expression Omnibus (GEO) website which is maintained by NCBI. I have not used it, but it comes with high expectations. If you have used it, please drop a line and I'll incorporate it into this short profile.
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This is the KEGG Pathway Database website, which is part of the Kyoto Encyclopedia of Genes and Genomes, and is a great database allowing you visualize heaps of different pathways. It's also interactive, so you can find a pathway (for eg) and then click on a protein or enzyme of your choice, and then get sequence info, what other pathways is it linked with, and other info depending on the type of protein it is. Simply put, I like this site.
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This is a link to the "Microarray Analysis Resources" website, which has a much more comprehensive list of microarray resources, including general databases, Analysis Tools, Functional Annotation (GO tools), Pathway Analysis and a few others. A good resource to help you analyze your data in general.
If you find another site that you have found helpful, please let me know and I will add it to this list. Hope this helps though!
____________________________________________________________
This is a link to the SOURCE website which allows you to input your gene (as a GenBank Accession number for eg) and get the gene name, symbol, ontologies, function, and several other potentially useful information. It is possible to do a single gene search, or a batch analysis. It is also possible to download your results as a tab-delimited .txt file.
____________________________________________________________
This is a link to the DAVID Bioinformatics website, which allows you to do similar things as the SOURCE website such as functional annotation. This site has several other useful features such as producing "Enrichment" scores, which, to my understanding, is an analysis of the likely-hood of a group of genes being found together. Or conversely, it gives you a numerical interpretation as to the likely-hood that these genes would come up together in a list at random. The results that this program produces all have links as well as many other useful statistical analysis. Well worth a look. (Performs an EASE analysis)
____________________________________________________________
This is another DAVID analysis website which allows you to post in gene lists from microarray experiments (or other experiment producing lists of genes) and results in sorting them into different functional categories based on their GO (Gene Ontology) Terms. It produces Annotation charts, GO Charts, Kegg Charts, Domain Charts, and provides access to EASE online (once you submit a gene list). You can also download EASE to run on your own computer from this site by following the links. I gathered alot of useful information from this website, and recommend it.
____________________________________________________________
The Gene Expression Omnibus (GEO) website which is maintained by NCBI. I have not used it, but it comes with high expectations. If you have used it, please drop a line and I'll incorporate it into this short profile.
____________________________________________________________
This is the KEGG Pathway Database website, which is part of the Kyoto Encyclopedia of Genes and Genomes, and is a great database allowing you visualize heaps of different pathways. It's also interactive, so you can find a pathway (for eg) and then click on a protein or enzyme of your choice, and then get sequence info, what other pathways is it linked with, and other info depending on the type of protein it is. Simply put, I like this site.
__________________________________________________________
This is a link to the "Microarray Analysis Resources" website, which has a much more comprehensive list of microarray resources, including general databases, Analysis Tools, Functional Annotation (GO tools), Pathway Analysis and a few others. A good resource to help you analyze your data in general.
