Instructions
	1. Summary 2. Usage 3. Output 4. References
	1. Summary Given the amino acid sequence of a putative transmembrane (TM) helix, the server gives a prediction of the corresponding apparent free energy difference, ΔGapp, for insertion of this sequence into the Endoplasmic Reticulum (ER) membrane by means of the Sec61 translocon. In principle, a negative value of ΔGapp indicates that the sequence is predicted to be recognized as a TM helix by the Sec translocon and integrated into the membrane. On the other hand, a positive value of ΔGapp does not necessarily imply that the sequence is not transmembrane (see below). The predictions are based on experimental results from [1] and [2] where systematically designed 19-residue long amino acid sequences have been expressed and tested for TM insertion using an in vitro assay. From these results, a quantitative model, described in [1], has been developed based on the assumption that amino acid contributions to ΔGapp are roughly additive, with additional terms to account for the hydrophobic moment and length of the helix respectively. ΔGapp is calculated as where l is the length of the segment, ΔGaa(i)app is the contribution from amino acid aa in position i, c0 is a weight parameter for the hydrophobic moment, and c1, c2 and c3 are parameters for describing helix length. One notable feature of the model is that amino acid contributions to ΔGapp are assumed to be position dependent, i.e. the free energy cost of inserting e.g. a charged amino acid near the middle of the membrane is higher than towards the interfacial region. 2. Usage The prediction server runs in two different "modes", for two different types of queries: ΔG prediction. Use this mode if you have the sequence of a potential TM helix for which you want to predict ΔGapp for membrane insertion. ΔGapp is calculated according to the equation above. Due to computational limitations, maximum 1000 sequences are allowed in each query. Example input: IWLWIGTIGMTLGTLYFVGR FYIITIFITTIAAAMYFAMATGF ALTIYWARYADWLFTTPLLLLDLSLLA TIATLIGLDVFMIGTGAIAALS TRIAWWAISTGALLALLYVLVGTL LFGRLRNLVIALWFLYPVVWILGT ILPLYWETAAFMVLDLSAKVGFGVILL Options: Length correction: Use the length correction described in [1] for helices of lengths other than 19 aa. Unchecking this box corresponds to setting parameters c1, c2 and c3 in the equation above equal to zero. (Default: Yes). Allow subsequence (if lower ΔG): When this box is checked, the query sequence is also searched for subsequences with lower predicted ΔGapp. Use this option if you are not sure about the helix boundaries. (Default: No). Full protein scan. Use this mode if you have a protein sequence that you want to search for putative TM helices. The server will search your sequence using a sliding window of variable lengths and output a graph displaying the predicted ΔGapp for transmembrane insertion throughout the length of the sequence, as well as the predicted TM segments. Sequences should be inserted in FASTA format. Due to computational limitations, maximum 10 protein sequences are allowed in each query. Example input: >O93740|BACR_HALS4 Bacteriorhodopsin - Halobacterium sp. (strain arg-4). MCCAALAPPMAATVGPESIWLWIGTIGMTLGTLYFVGRGRGVRDRKMQEFYIITIFITTI AAAMYFAMATGFGVTEVMVGDEALTIYWARYADWLFTTPLLLLDLSLLAGANRNTIATLI GLDVFMIGTGAIAALSSTPGTRIAWWAISTGALLALLYVLVGTLSENARNRAPEVASLFG RLRNLVIALWFLYPVVWILGTEGTFGILPLYWETAAFMVLDLSAKVGFGVILLQSRSVLE RVATPTAAPT Options: Helix min length: Minimum length of sliding window used for searching the sequence. (Default: 17; min value: 9). Helix max length: Maximum length of sliding window used for searching the sequence. (Default: 27; max value: 40). Length correction: Use the length correction described in [1] for helices of lengths other than 19 aa. Unchecking this box corresponds to setting parameters c1, c2 and c3 in the equation above equal to zero. (Default: Yes). Show graphics: Display a color graph with predicted ΔGapp against position in sequence, for different lengths of the sliding window. (Default: Yes). 3. Output The server outputs predicted ΔGapp values for the inserted sequences according to the equation above, and in case of the "full protein scan" mode (see above), also outputs a graph showing predicted ΔGapp values against position in sequence for different helix lengths. Please note: Even if the predicted ΔGapp value is positive, that does not necessarily imply that the sequence entered can not be transmembrane. All it suggests is that the segment would not be inserted efficiently by itself, and may need stabilizing interactions from surrounding helices. As shown in figure 4 in [1], a large fraction of known TM helices from multi-spanning membrane protein structures have predicted ΔGapp above zero. 4. References When using this server to predict ΔGapp for published work, please cite: [1] Hessa, T., Meindl-Beinker, N., Bernsel, A., Kim, J., Sato, Y., Lerch, M., Lundin, C., Nilsson, I., White, SH. and von Heijne, G. (2007) Molecular code for transmembrane-helix recognition by the Sec61 translocon. Nature. 450, 1026-1030. [PubMed] In addition, when referring to the experimental ΔGapp measurements, please also cite: [2] Hessa, T., Kim, H., Lundin, C., Boekel, J., Andersson, H., Nilsson, I., White, SH. and von Heijne, G. (2005) Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433, 377-381. [PubMed]

	# Helices predicted: