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Open Access Highly Accessed Research article

Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis

Tobias H Klöpper1, Nickias Kienle2, Dirk Fasshauer2 and Sean Munro1*

Author Affiliations

1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK

2 Department of Cellular Biology and Morphology, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland

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BMC Biology 2012, 10:71  doi:10.1186/1741-7007-10-71

Published: 8 August 2012

Additional files

Additional file 1:

Overview of Rabs in representative species outside of opisthokonts. Species from various phyla are as indicated and where there is presence of at least one Rab of the given type, this is indicated by a cross. The species are from the four eukaryotic supergroups, S+C, SAR+CCTH; A, Archaeplastida; Ex, Excavata; Unik, Unikonta. Format: PDF. Size: 94 kb.

Format: PDF Size: 88KB Download file

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Open Data

Additional file 2:

Statistical validation of Rab classifications. (A) To assess the quality of our hidden Markov models (HMMs), a resampling method was used, and 90% of the sequences used to generate each model were randomly gathered, and new models generated from these. The other Rabs were used as the search database, with a fixed size of the database of 100,000 sequences. The profile with the best expectation value was assumed to be the correct class, and the resampling was repeated 1000 times. For each model, the PPR (grey, left) and the sensitivity (black, right) are shown. All models achieved at least 95% PPR and sensitivity. False positives and false negatives occurring within the analysis were inspected and it was found that for a number of cases, the wrong classification was caused by metazoan-specific duplications. For example, Rab10 had the lowest observed sensitivity (95.4%), but on further investigation all false negatives were identified as Rab8 (42% of the false positives). Because Rab10 is a metazoan duplication of Rab8, the classification is not exactly wrong but is rather inaccurate. The metazoan models are likely to improve once further metazoan genomes become available. (B-D) As Rab proteins are members of the larger Ras protein family we needed to also address the problem of randomly identifying non-Rab sequences with the models. Because the models were generated using Rab sequences, non-Rab sequences should show a weaker fit to the model. To determine how specific the models were for Rab proteins, approximately 2000 members of the larger Ras family (for example, Arf, Arl, Rho and Ran proteins) were selected, and each of the models was used to predict 'Rab' motifs in these non-Rab sequences. We visualized the results using box plots with the 5% and 95% percentiles, shown as whiskers. Plots show the scores from sequences that were accepted to be members of the family modeled (B), or from the set of non-Rab sequences that we selected (D). For both graphs, the negative logarithm of the expectation value achieved by the motifs was plotted. The difference in e-value distribution between the two datasets was then used to define cut-offs for the confidence of our predictions. The first 'strict' cut-off was generated using the 5% percentile of the expectation value distribution in (B), and the second 'soft' cut-off was generated using the 95% percentile of the expectation value distribution from (D). Values are displayed in (C). Format: PDF Size: 1.6 mb.

Format: PDF Size: 1.6MB Download file

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Open Data

Additional file 3:

Overview of the species used to generate Rab-specific HMMs. Species and species subsets used in computations and figures, showing species name, the abbreviation used for the species, the source database from which the sequences were obtained and the set in which it was used: models for the LECA groups, models for opisthokont groups, and models for metazoan-specific groups. Format: XLS Size: 29 kb.

Format: XLS Size: 28KB Download file

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Open Data

Additional file 4:

Alignment of group I Rab proteins in FASTA format. Format: TXT Size: 147 kb.

Format: TXT Size: 140KB Download file

Open Data

Additional file 5:

Alignment of group II Rab proteins in FASTA format. Format: TXT Size: 70 kb.

Format: TXT Size: 64KB Download file

Open Data

Additional file 6:

Alignment of group III Rab proteins in FASTA format. Format: TXT Size: 53 kb.

Format: TXT Size: 49KB Download file

Open Data

Additional file 7:

Alignment of group IV Rab proteins in FASTA format. Format: TXT Size: 86 kb.

Format: TXT Size: 84KB Download file

Open Data

Additional file 8:

Alignment of group V Rab proteins in FASTA format. Format: TXT Size: 25 kb.

Format: TXT Size: 22KB Download file

Open Data

Additional file 9:

Alignment of group VI Rab proteins in FASTA format. Format: TXT Size: 16 kb.

Format: TXT Size: 13KB Download file

Open Data