\documentclass[10pt,letterpaper]{article}

\usepackage{xcite}
\usepackage{xr-hyper}
\usepackage[draft]{hyperref}
\externaldocument[M-]{protrusion_ms}
\externalcitedocument{protrusion_ms}

%\usepackage{color}
\usepackage{url}
\usepackage[font={scriptsize}]{caption}
\usepackage{graphicx}
\usepackage{array}
\newcommand{\note}[1]{\textcolor{red}{#1}}

\def\inc/{\note{(cite)}}
\def\inr/{\note{(ref)}}
\def\ea/{\textit{et al.}}
\def\ca/{$\textrm{C}_{\alpha}$}
\def\cb/{$\textrm{C}_{\beta}$}

\newcommand*{\expr}[1]{\textcolor{red}{#1}}
\newcommand*{\predr}[1]{{#1}}
\newcommand*{\bothr}[1]{\textbf{#1}}

\def\periphcolor/{blue}
\def\tmcolor/{red}
\def\matmet/{\emph{Materials and methods}}

\def\colwidth{6cm}
\def\doublewidth{12cm}
\def\smallwidth{3.5cm}

\renewcommand*{\thefigure}{S\arabic{figure}}
\renewcommand*{\thetable}{S\arabic{table}}
\renewcommand*{\thesection}{S\arabic{section}}
\newcommand*{\reanalysisCaption}[1]{See caption of corresponding Figure~\ref{#1} in main text.}

\pdfsuppresswarningpagegroup1

\title{A model for hydrophobic protrusions on peripheral membrane proteins - Supporting information}
\author{Edvin Fuglebakk and Nathalie Reuter}
\begin{document}

\newcommand{\FigTmPeriphAaComparison}{
\begin{figure}[t]
\centering
 \includegraphics[width=\colwidth]{FigTmPeriphAaComparisonAlternative.pdf}

\caption{Panel A shows the weighted fractions of hydrophobic amino acids on protrusions from the set \emph{Peripheral-P} proteins (\periphcolor/) and from proteins in the \emph{Reference proteins} (\tmcolor/). In panel B, the contrast between the two sets is quantified by the odds ratio, so that positive values reflect higher frequencies in the set of peripheral proteins than in the reference set. \reanalysisCaption{M-FigTmPeriphAaComparison}}
\label{FigTmPeriphAaComparisonRe}
\end{figure}
}

\newcommand{\FigNeighboursVsOddsRatio}{
	\begin{figure}[t]
		\captionsetup{width=\colwidth}
		\centering
		\includegraphics[width=\colwidth]{FigNeighboursVsOddsRatioAlternative.pdf}
		\caption{The plot shows the logarithm of the odds-ratio comparing the frequency of hydrophobes on \emph{vertex} residues in the set \emph{Peripheral-P} and the \emph{Reference proteins}. Positive values reflect higher frequencies in the peripheral proteins. \reanalysisCaption{M-FigNeighboursVsOddsRatio}}
		\label{FigNeighboursVsOddsRatio}
	\end{figure}
}

\newcommand{\FigSSE}{
	\begin{figure}[t]

		\centering
		\includegraphics[width=\colwidth]{FigSSEAlternative.pdf}

	\caption{Panel A shows the weighted number of \emph{protruding hydrophobes} associated with the different types of secondary structure elements. We have differentiated between protrusions that have at least one co-insertable protruding hydrophobe (right, labeled ``Co-ins.''), and those that have not (left, labeled ``Isolated''). Panel B compares the weighted frequencies of hydrophobes on protruding secondary structures between the set \emph{Peripheral-P} and the \emph{Reference proteins}, using the odds ratio. Positive values reflect higher frequencies in the peripheral proteins.\reanalysisCaption{M-FigSSE}}
	\label{FigSSE}
	\end{figure}
}


\date{}
\maketitle
\clearpage

\section{Data sets of proteins with experimentally verified membrane-binding sites}
We used a small set of protein structures to establish the definition of protrusions and adjust the parameters $c$ and $n$ (Cf. \matmet/). The dataset consists of structures of peripheral proteins with striking protrusions at their experimentally-verified membrane-binding site. Table~\ref{tab:manualProtrusions} contains the list of PDB codes, the protein family to which they belong and the amino acids forming the membrane-binding site, along with some comparisons with predicted binding (\emph{Likely inserted hydrophobes}) sites not presented in the manuscript.

We also collected a larger dataset of peripheral proteins with experimentally-identified binding sites. The structures and binding sites are listed in Table~\ref{tab:verifiedBinders}, along with comparisons with our predictions (\emph{Likely inserted hydrophobes}). This dataset does not overlap with the one listed in Table~\ref{tab:manualProtrusions} and could thus be used for analysis purposes, and in particular for those results reported in Figure~\ref{M-FigCompExperiment} of the main manuscript. This set has some overlap with the list provided by Lomize \ea/~\cite{Lomize:2007gm}.

The proteins presented in Figure~\ref{M-FigDomainExamples} are taken from both of these sets. For convenience we have compiled a separate table, repeating the information about binding sites and comparisons with our predictions in Table~\ref{tab:domainProtrusions}.

\begin{table}
		\small
	\begin{tabular}{c p{3cm} p{1cm} p{3cm} p{1cm} p{4cm}}
		PDB ID & interfacial binding site & LIH & co-insertables & angle & family\\
		\hline
		1RLW & F35, M38, L39, N95, Y96, V97, M98~\cite{Malmberg:2003uk} & \textbf{F35} & \textbf{M98} \textbf{M38} \textbf{L39} & 19.2 & C2-domain\\
		1BYN & M173, G174, R233, F234, K235 \cite{Frazier:2003wf, Gerber:2002fp} & \textbf{M173} & \textbf{F234} & 18.2 & C2-domain\\
		1UOV & V304, G305, I367, K369~\cite{Rufener:2005ie} & L270 & & 72.4 & C2-domain\\
		1GMI & I89, Y91~\cite{CorbalanGarcia:2003ba} & \textbf{I89} & & 6.8 &C2-domain\\
		1H6H & F35, Y94, V95~\cite{Stahelin:2003cv} & \textbf{Y94} & \textbf{F35} & 23.7 & PX domain\\
		1CZS & W26, W27~\cite{Kim:2000em} & \textbf{W26} & \textbf{W27} L79 & 4.3 & Discodin domain\\
		1D7P & M2199, F2200, L2251, L2252~\cite{Gilbert:2002ja} & \textbf{L2251} & \textbf{M2199} \textbf{L2252} \textbf{F2200} & 18.9 & Discodin domain\\
		1T6M & W51, Y92, Y208, W246, Y250, Y252~\cite{Feng:2003ik, Feng:2002fc, Grauffel:2013uf} & \textbf{W246} & I47 & 8.5 & Bacterial PLC\\
		1H0A & L6, M10~\cite{Stahelin:2003is} & I13 & \textbf{L6} & 20.4 & ENTH domain\\
		%1Q4G & I74, W75, W77, L78, F88, F91, L92, W98,L99, F102~\cite{Spencer:1999df} & \\
		1LOX & L195~\cite{Walther:2004ut} & L71 & Y292 F70 L192 \textbf{L195} & 20.5 & Lipoxygenases\\
	\end{tabular}
	\caption{Peripheral protein structures used for defining and parameterizing the model of hydrophobic protrusions. Family classifications are taken from OPM\cite{Lomize:2011iz}. Although these proteins have not been included in Figure~\ref{M-FigCompExperiment}, we list here the \emph{Likely inserted hydrophobe} (LIH), and protruding hydrophobes co-insertable with it. The \textit{angle} column informs about the comparisons of orientations between our prediction and OPM (similar to those presented in Figure~\ref{M-FigCompExperiment}, as given by Eq~\ref{M-eq:orientation}).}
	\label{tab:manualProtrusions}
\end{table}

\begin{table}
	\small
	\begin{tabular}{c p{3cm} p{1cm} p{3cm} p{1cm} p{4cm}}
		PDB ID & interfacial binding site & LIH & co-insertables & angle & classification\\
		\hline
		1DSY & M186 N189 R216 R249 R252~\cite{Kohout:2003bs} & M269 & & 166.8 & C2-domain\\
		1O7K & R43 I65 W80~\cite{Stahelin:2003cv} & L35 & & 166.5 & PX domain\\
		1HYJ & V21 T22~\cite{Kutateladze:2001ir} & W3 & & 94.9 & FYVE PIP$_{3}$ domain\\
		1VFY & L185 L186 R193~\cite{Stahelin:uj} & \textbf{L186} & \textbf{L185} & 14.7 & FYVE PIP$_{3}$ domain\\
		1PTR & L250 W252 L254~\cite{Wang:2001jw} & \textbf{L254} & M239 & 24.2 & C1 domain\\
		1A8A & T72 S144 W185 S228 S303~\cite{BCampos:1998fa} & L29 & \textbf{W185} & 72.2 & Annexins \\
		1DM5 & E142 S144 G145~\cite{Isas:2004fw} & L101 & L260 I29 I185 & 49.2 & Annexins \\
		1IAZ & W112 W116~\cite{Anderluh:2005kk} & \textbf{W112} & & 6.3 & Pore-forming Equinatoxin\\
		1NB1 & C1 G2 E4 T5 V6 G7 S18 W19 P20 V21 C22 G26 L27 P28 V29~\cite{Shenkarev:2006fg} & \textbf{L27} & \textbf{W19} & 77.4 & Cyclotide\\
		1POC & I2 K14 I78~\cite{Lin:1998uz} & L90 & \textbf{I78} I1 & 71.2 & Insect sec. PLA$_{2}$\\
		1N28 & V3 K10 L19 F23 F63 K115~\cite{Canaan:2002dp} & \textbf{F63} & & 65.1 & Vertebrate sec. PLA$_{2}$\\
		1POA & W61 F64 Y110~\cite{Stahelin:2001cr} & W19 & \textbf{F64} Y3 & 74.8 & Vertebrate sec. PLA$_{2}$\\
		1VAP & W20 W109~\cite{Lathrop:2001wu} & F3 & M61 L19 & 90.0 & Vertebrate sec. PLA$_{2}$\\
		4P2P & W3~\cite{Tatulian:2005ja} & L19 & \textbf{W3} M20 & 54.2 & Vertebrate sec. PLA$_{2}$\\
		1COY & M81~\cite{Chen:2000vf} & M332 & L369 W333 Y437 & 28.5 & GMC oxidoreductases\\
		1PFO & W464 W466~\cite{SekinoSuzuki:1996uy} & L491 & \textbf{W466} Y492 L462 & 14.4 & Chol.-dep. Cytolysin \\
		1D1H & W30~\cite{Phillips:2005gn} & F6 & \textbf{W30} & 79.1 & Spider toxins \\
		1PXQ & W34~\cite{Thennarasu:2005jw} & W34 & & 0.0 & Subtilosin A\\
		2FNQ & W413 W449\cite{Oldham:2005fb} & Y448 & L514 \textbf{W449} F414 W413 & 12.6 & Lipoxygenases\\
		1G13 & T90 L126 N136~\cite{Mathias:2009bj} & W131 & I162 & 51.0 & ML domain\\
		1EIN & P42 D96 T123 I252~\cite{Hedin:2002eu} & \textbf{I252} & I255 I86 L93 L227 & 50.9 & Fungal lipases\\
		3PAK & Y164 R216 Y221 R222~\cite{Goh:2016hv} & L219 & & 68.4 & Lectin domain\\
		1F6S & K98 V99~\cite{Agasoster:2003bja} & & & & C-type lysozyme\\
		2DA0 & K18 K19 I23 K25 N30 N48 N77~\cite{Jian:2015do} & \textbf{I23} & & 14.0 & Pleckstrin-homology d.\\
	\end{tabular}
	\caption{Protein structures and corresponding membrane-binding sites used for systematic comparison with the \textit{Likely Inserted Hydrophobe} (LIH). Protruding hydrophobes that are co-insertable with the LIH is listed in the column co-insertables. The \textit{angle} column informs about the comparisons of orientations between our prediction and OPM (similar to those presented in Figure~\ref{M-FigCompExperiment}). Family classifications are from OPM~\cite{Lomize:2011iz}, except for 3PAK and 1F6S which were taken from SCOPe~\cite{Fox:2014vx} as the structures are not present in OPM. Quaternary structures are also taken from OPM, except for 3PAK and 1F6S; those were obtained from the literature. Residue numbering corresponds to that used in the listed PDB ID. All structures are either monomers or homo-oligomers where all chains are equally likely to interact with the membrane. Chain identifiers are therefore not provided.}
	\label{tab:verifiedBinders}
\end{table}

\begin{table}
		\small
	\begin{tabular}{c p{3cm} p{1cm} p{3cm} p{1cm} p{4cm}}
		PDB ID & interfacial binding site & LIH & co-insertables & angle & family\\
		\hline
		1RLW (A) & F35, M38, L39, N95, Y96, V97, M98~\cite{Malmberg:2003uk} & \textbf{F35} & \textbf{M98} \textbf{M38} \textbf{L39} & 19.2 & C2-domain\\
		1H6H (B) & F35, Y94, V95~\cite{Stahelin:2003cv} & \textbf{Y94} & \textbf{F35} & 23.7 & PX domain\\
		1POA (C) & W61 F64 Y110~\cite{Stahelin:2001cr} & W19 & \textbf{F64} Y3 & 74.8 & Vertebrate sec. PLA$_{2}$\\
		1PTR (D) & L250 W252 L254~\cite{Wang:2001jw} & \textbf{L254} & M239 & 24.2 & C1 domain\\
		1H0A (E) & L6, M10~\cite{Stahelin:2003is} & I13 & \textbf{L6} & 20.4 & ENTH domain\\
		1VFY (F) & L185 L186 R193~\cite{Stahelin:uj} & \textbf{L186} & \textbf{L185} & 14.7 & FYVE PIP$_{3}$ domain\\
	\end{tabular}
	\caption{Binding sites of common membrane-binding domains presented in Figure~\ref{M-FigDomainExamples}. All of these proteins are also listed in Tables~\ref{tab:manualProtrusions} and~\ref{tab:verifiedBinders}, and the table format is specified there. They are repeated here for ease of comparison with Figure~\ref{M-FigDomainExamples}, and the corresponding panel in that figure is indicated in parenthesis in the column PDB ID.}
	\label{tab:domainProtrusions}
\end{table}


\clearpage

\section{Additional analysis performed}
In the manuscript we have presented analysis on two pairs of data sets for analysis that aim to contrast surface properties between peripheral membrane proteins and other proteins. For analysis that aim to characterize protrusions on peripheral proteins, we have chosen to present these results only for one of the pairs. This is because we consider this a better representation of peripheral membrane binders, as quaternary structure has been more carefully scrutinized. Also, one of these analysis can only be done for the set \emph{Peripheral}, namely the comparison with the OPM-database~\cite{Lomize:2011iz} presented in the manuscript (Figure~\ref{M-FigLargestFacetLocation}). We present in this supplementary material(Figures~\ref{FigNeighboursVsOddsRatio},~\ref{FigTmPeriphAaComparisonRe} and~\ref{FigSSE}), analysis of the sets \emph{Peripheral-P} and \emph{Reference proteins}, corresponding to the analysis of the sets \emph{Peripheral} and \emph{Non-binding surfaces} presented in Figures~\ref{M-FigNeighboursVsOddsRatio}, \ref{M-FigTmPeriphAaComparison} and~\ref{M-FigSSE}. The conclusions drawn from the primary datasets are supported by the analysis of \emph{Peripheral-P} and \emph{Reference proteins}. The relative importance of large aliphatic residues on protruding locations in peripheral proteins is reproduced (Figure~\ref{FigTmPeriphAaComparisonRe}). There is still a stronger contrast between the data sets when the analysis is restricted to vertex residues of low protein density (Figure~\ref{FigNeighboursVsOddsRatio}). The analysis of secondary structure elements also yields a result similar to what was obtained for the primary datasets (Figure~\ref{FigSSE}).

\FigNeighboursVsOddsRatio
\FigTmPeriphAaComparison
\FigSSE

\section*{References}
See main text

%\bibliographystyle{unsrt}
%\bibliography{}

\end{document}