MOTIVATION: Membrane proteins are clinically relevant, yet their crystal structures are rare. Models of membrane proteins are typically built from template structures with low sequence identity to the target sequence, using a sequence-structure alignment as a blueprint. This alignment is usually made with programs designed for use on soluble proteins. Biological membranes have layers of varying hydrophobicity, and membrane proteins have different amino-acid substitution preferences from their soluble counterparts. Here we include these factors into an alignment method to improve alignments and consequently improve membrane protein models. RESULTS: We developed Membrane Protein Threader (MP-T), a sequence-structure alignment tool for membrane proteins based on multiple sequence alignment. Alignment accuracy is tested against seven other alignment methods over 165 non-redundant alignments of membrane proteins. MP-T produces more accurate alignments than all other methods tested (δF(M) from +0.9 to +5.5%). Alignments generated by MP-T also lead to significantly better models than those of the best alternative alignment tool (one-fourth of models see an increase in GDT_TS of ≥4%). AVAILABILITY: All source code, alignments and models are available at http://www.stats.ox.ac.uk/proteins/resources

MOTIVATION: Membrane proteins are both abundant and important in cells, but the small number of solved structures restricts our understanding of them. Here we consider whether membrane proteins undergo different substitutions from their soluble counterparts and whether these can be used to improve membrane protein alignments, and therefore improve prediction of their structure. RESULTS: We construct substitution tables for different environments within membrane proteins. As data is scarce, we develop a general metric to assess the quality of these asymmetric tables. Membrane proteins show markedly different substitution preferences from soluble proteins. For example, substitution preferences in lipid tail-contacting parts of membrane proteins are found to be distinct from all environments in soluble proteins, including buried residues. A principal component analysis of the tables identifies the greatest variation in substitution preferences to be due to changes in hydrophobicity; the second largest variation relates to secondary structure. We demonstrate the use of our tables in pairwise sequence-to-structure alignments (also known as 'threading') of membrane proteins using the FUGUE alignment program. On average, in the 10-25% sequence identity range, alignments are improved by 28 correctly aligned residues compared with alignments made using FUGUE's default substitution tables. Our alignments also lead to improved structural models. AVAILABILITY: Substitution tables are available at: http://www.stats.ox.ac.uk/proteins/resources.