figures_and_tables_paper.Rmd

---
lang: en-gb
title: "Implications of kappa-casein evolutionary diversity for the self-assembly and stabilization of casein micelles: figures and tables"
author: "Jean Manguy, Denis C. Shields"
output: 
  html_document:
    toc: true
    toc_depth: 1
    toc_float: true
    code_folding: show
    mathjax: null
    self_contained: yes
    dev: png
---

```{r setup, include=FALSE}
knitr::opts_chunk$set(
  echo = TRUE,
  fig.align = "center",
  dev.args = list(png = list(type = "cairo"))
)

title <- "Implications of kappa-casein evolutionary diversity for the self-assembly and stabilization of casein micelles"
authors <- "Jean Manguy and Denis C. Shields"
year <- "2019"
journal <- "Royal Society Open Science"
doi_article <- "10.1098/rsos.190939"
doi_zenodo <- "10.5281/zenodo.2587122"
```


`r badger::badge_custom("R ≥", "3.5", color = "green")`
`r badger::badge_custom("Python ≥", "3", color = "green")`
`r badger::badge_doi(doi_zenodo, "blue")`
`r badger::badge_doi(doi_article, "red")`


```{r load-project, message=FALSE, warning=TRUE, echo=FALSE}
library(ProjectTemplate)
load.project()
plot_output_dir <- "graphs"
table_output_dir <- "tables"
walk(c(file.path(plot_output_dir), file.path(table_output_dir)), dir.create, showWarnings = FALSE)
```

# Functions and variables

## misc

```{r misc}
underscore_to_space <- partial(str_replace_all, pattern = "_", replacement = " ")
oldest_node_tree <- trimmed_species_tree_as_df %>% pull(x) %>% max()

# labels for plot axis
max_msa_index <- kappa_caseins_alignment_positions %>% pull(msa_index) %>% max()
align_breaks <- c(
  1, # min
  max_msa_index, # max
  seq(100, max_msa_index, 100) # intermediate positions
)
align_breaks_labels <- c(
  1, # min
  max_msa_index, # max
  seq(100, max_msa_index, 100) # intermediate positions
) %>% as.character()
align_minor_breaks <- c(1, max_msa_index, seq(25, max_msa_index, 25))
```

## export plots and tables


```{r exportfunctions}
save_plot_pdf <- partial(
  ggsave,
  path = plot_output_dir,
  device = cairo_pdf,
  units = "in"
)
save_plot_jpg <- partial(
  ggsave,
  path = plot_output_dir,
  dpi = "retina",
  units = "in"
)

save_plot_tiff <- partial(
  ggsave,
  path = plot_output_dir,
  dpi = "retina",
  units = "in"
)

save_plot <- function(plot, name, ...) {
  suppressMessages(
    save_plot_pdf(plot = plot, filename = glue("{name}.pdf"), ...)
  )
  suppressMessages(
    save_plot_jpg(plot = plot, filename = glue("{name}.jpg"), ...)
  )
  suppressMessages(
    save_plot_tiff(plot = plot, filename = glue("{name}.tiff"), ...)
  )
  invisible(plot)
}
save_table <- function(table, name, ...) {
  write(x = table, file = file.path(table_output_dir, glue("{name}.tex")), ...)
  invisible(plot)
}
```

## colours

Custom amino acid colours based on the colourblind friendly OkabeIto colour palette (see http://jfly.iam.u-tokyo.ac.jp/color/ and https://github.com/clauswilke/colorblindr) 

```{r coloursaa}
aa_custom_OkabeIto_colours <- c(
  "cysteine" = "#E69F00", # orange
  "histidine" = "#56B4E9", # light blue
  "charged +" = "#0072B2", # marine blue
  "charged -" = "#D55E00", # red
  "aromatic" = "#CC79A7", # pink
  "proline" = "#F0E442", # yellow
  "polar" = "#009E73", # green
  "apolar" = "#999999", # light grey
  "glycine" = "#999999", # light grey
  "gap" = "#ffffff" # white
)
```

Kappa-casein parts colours

```{r coloursparts}
colour_parts_kappa_casein <- c(
  "PKC" = "#e69f00", # orange
  "GMP" = "#56b4e9" # blue
)
```


## Base plots

```{r base_plot_alignment_parts, cache=TRUE}
# scales and theme ----
scale_x_align_residue_plot <- scale_x_continuous(
  expand = c(0, 0),
  breaks = c(align_breaks),
  labels = c(align_breaks_labels),
  minor_breaks = align_minor_breaks
)

scale_x_align_residue_plot_pkc_gmp <- scale_x_continuous(
  expand = c(0, 0),
  breaks = c(align_breaks, position_align_chymosin_cleavage_site),
  labels = c(align_breaks_labels, "PKC/GMP"),
  minor_breaks = align_minor_breaks
)

scale_y_align_residue_plot <- scale_y_continuous(
  expand = c(0, 0),
  breaks = c(0, 0.5, 1)
)




# base plots for figures and some supplementary figures
plot_kappa_caseins_parts <- kappa_caseins_alignment_positions_parts %>%
  ggplot(mapping = aes(x = msa_index, y = species, fill = part)) +
  geom_tile(size = 0) + # 0 for no border
  scale_fill_manual(values = c(colour_parts_kappa_casein), na.value = "white", guide = guide_legend(reverse = TRUE)) +
  scale_x_align_residue_plot +
  scale_y_discrete(
    position = "right",
    labels = underscore_to_space,
    expand = c(0, 0)
  ) +
  theme_bw() +
  theme(
    text = element_text(colour = "black", family = "Arial"),
    axis.text = element_text(colour = "black", family = "Arial"),
    axis.ticks.y = element_line(colour = "black"),
    axis.ticks.x = element_line(colour = "black"),
    # legend.title = element_blank(),
    # legend.position = c(0, 1),
    # legend.justification = c(0, 1),
    # legend.background = element_rect(fill = "transparent"),
    # legend.box.background = element_rect(colour = "black", fill = "white", size = 0.2),
    # legend.spacing = unit(1, "mm"),
    # legend.key = element_blank(),
    # legend.box.spacing = unit(0.01, "line"),
    # legend.key.size = unit(5, "pt"),
    # legend.text = element_text(margin = margin(0, 0, 0, 2), size = 8),
    # legend.margin = margin(1, 1, 1, 1),
    axis.ticks = element_line(size = 0.2),
    axis.text.y = element_text(size = 7, face = "italic"),
    axis.title.x = element_text(size = 8),
    axis.text.x = element_text(size = 7),
    panel.background = element_rect(fill = "white", colour = "white"),
    plot.background = element_rect(fill = "white", colour = "white"),
    panel.border = element_rect(fill = "transparent", colour = "black", size = 0.2),
    axis.title.y = element_blank(),
    plot.title = element_text(size = 9),
    plot.subtitle = element_blank(),
    panel.grid.major.x = element_line(size = 0.1),
    panel.grid.major.y = element_line(size = 0.1),
    panel.grid.minor.x = element_blank()
  ) +
  labs(x = "aligned amino acid position") +
  NULL

# equal expand values for the y axis to assure the 2 plots align well
indels_kappacasein_parts_plot_expand <- c(0.005, 0.0)


# species tree on the left
plot_kappa_casein_tree <- ggplot(trimmed_species_tree, size = 0.5) +
  geom_tree(size = 0.2) +
  geom_treescale(x = 10, y = 40, fontsize = 2.5, linesize = 0.4, offset = 1) +
  theme_bw() +
  theme(
    text = element_text(colour = "black", family = "Arial"),,
    plot.margin = margin(0, 0, 0, 0),
    panel.spacing = margin(0, 5, 5, 5),
    panel.border = element_blank(),
    panel.grid.major = element_blank(),
    panel.grid.minor = element_blank(),
    axis.text = element_blank(),
    axis.title = element_blank(),
    axis.ticks = element_blank(),
  ) +
  scale_y_continuous(expand = indels_kappacasein_parts_plot_expand) +
  scale_x_continuous(expand = c(0, 0)) +
  xlim_tree(167)
```


# Figure 1


```{r figure1, echo=TRUE, fig.height=4.1, fig.width=6, cache=TRUE}
# custom geom to be used for annotation with consistent style
geom_cladelabel_custom <- partial(
  geom_cladelabel,
  fontsize = 2.4,
  offset = 0.6,
  offset.text = 0.1,
  family = "arial",
  barsize = 0.4
)

# geological time periods
# package geoscale
# Gradstein, F. M., Ogg, J. M., and Schmitz, M., 2012, A geologic time scale, Boston, USA, Elsevier.
data(timescales)

mammals_periods <- timescales %>%
  purrr::pluck("ICS2015") %>%
  filter(End < oldest_node_tree, !is.na(Part_of)) %>%
  rename(geologic.period = Part_of) %>%
  group_by(geologic.period) %>%
  summarise(start = max(Start), end = min(End)) %>%
  ungroup() %>%
  mutate(geologic.period = fct_reorder(geologic.period, end)) %>%
  arrange(desc(geologic.period)) %>%
  identity()

mammals_periods_breaks <- c(mammals_periods$start) %>% round(1)

# Panel A - maximum likelihood protein tree
plot_fitted_tree <- fitted_tree %>%
  ggtree(size = 0.1, color = "black") +
  xlim_tree(3.4) +
  geom_treescale(
    x = 0.1,
    y = 30,
    offset = 2,
    fontsize = 2.5,
    linesize = 0.4,
    width = 0.1
  ) +
  geom_tiplab(
    size = 0.7,
    mapping = aes(label = underscore_to_space(label)),
    family = "arial",
    fontface = "italic"
  ) +
  coord_cartesian(clip = "off") +
  scale_x_continuous(expand = c(0.02, 0)) +
  scale_y_continuous(expand = c(0.02, 0)) +
  geom_cladelabel_custom(node = 197, label = "Monotremes") +
  geom_cladelabel_custom(node = 196, label = "Marsupials") +
  geom_cladelabel_custom(node = 194, label = "Afrotheria") +
  geom_cladelabel_custom(node = 106, label = "Glires") +
  geom_cladelabel_custom(node = 186, label = "Bats") +
  geom_cladelabel_custom(node = 139, label = "New world monkeys") +
  geom_cladelabel_custom(node = 126, label = "Old world monkeys") +
  geom_cladelabel_custom(node = 134, label = "Apes") +
  geom_cladelabel_custom(node = 175, label = "Carnivorans") +
  geom_cladelabel_custom(node = 149, label = "Ruminantia") +
  geom_cladelabel_custom(node = 164, label = "Cetaceans") +
  geom_cladelabel_custom(node = 169, label = "Camelids") +
  geom_cladelabel_custom(node = 172, label = "Odd-toed ungulates") +
  labs(
    tag = "A"
  ) +
  NULL



# Panel B- scatter plot
plot_compare_evodist_divtime <- compare_evodist_divtime %>%
  filter(corrected == TRUE) %>%
  ggplot(
    mapping = aes(
      x = divergence.time,
      y = distance.fit,
      label = glue("{species.1}-{species.2}") # dev only
    )
  ) +
  geom_vline( # separates geological periods
    xintercept = mammals_periods_breaks,
    colour = "darkgrey",
    linetype = "dotted", size = 0.5
  ) +
  geom_point( # couples of species
    size = 0.7,
    alpha = 0.1,
    colour = "black"
  ) +
  geom_text( # annotate geological periods at the top
    data = mammals_periods %>% filter(geologic.period != "Quaternary"),
    mapping = aes(label = geologic.period, x = start + (end - start) / 2),
    y = Inf,
    family = "arial",
    vjust = 1.4,
    size = 2.5
  ) +
  scale_x_continuous(
    limits = c(0, max(mammals_periods_breaks)),
    minor_breaks = seq(0, 200, 10),
    sec.axis = sec_axis(~., breaks = mammals_periods_breaks),
    expand = c(0.01, 0)
  ) +
  labs(
    x = "Divergence Time (Mya)",
    y = "Pairwise distance",
    tag = "B"
  ) +
  theme_bw() +
  theme(
    legend.position = "none",
    axis.text = element_text(size = 7),
    axis.title = element_text(size = 9),
    axis.ticks.x = element_line(size = 0.2),
    panel.border = element_rect(fill = NA, colour = "black", size = 0.4)
  ) +
  NULL



(plot_compare_evodist_divtime_combined <- plot_fitted_tree +
  plot_compare_evodist_divtime +
  plot_layout(widths = c(2.8, 5.2)) &
  theme(
    text = element_text(colour = "black", family = "Arial", face = "plain")
  )
)


save_plot(
  plot = plot_compare_evodist_divtime_combined,
  name = "figure_1"
)
```

# Figure 2



```{r figure2, fig.height=8.25, fig.width=6, cache=TRUE}
plot_indels_kappacasein_parts <- plot_kappa_caseins_parts +
  geom_rect( # PTRs annotations
    data = df_plot_indels,
    mapping = aes(
      ymin = as.numeric(species) - 0.4,
      ymax = as.numeric(species) + 0.4,
      xmin = N_flank_position - 1,
      xmax = C_flank_position,
      colour = type
    ),
    inherit.aes = FALSE,
    alpha = 0.0,
    size = 0.25
  ) +
  scale_colour_manual(values = c("tandem repeat" = "black")) +
  theme(
    text = element_text(colour = "black", family = "Helvetica"),
    legend.position = "top",
    legend.justification = c(0, 1),
    axis.title.y = element_blank(),
    axis.line.y = element_blank(),
    axis.text.y = element_text(size = 6, face = "italic", colour = "black"),
    axis.ticks = element_line(size = 0.2),
    legend.title = element_text(size = 8),
    legend.text = element_text(size = 8),
    plot.margin = margin(0, 0, 0, 0),
    panel.spacing = margin(0, 5, 5, 0),
    legend.background = element_rect(fill = "transparent"),
    # legend.box.background = element_rect(debug = TRUE),
    # legend.spacing = unit(0, "mm"),
    # legend.key = element_blank(),
    panel.background = element_rect(fill = "white", colour = "white"),
    plot.background = element_rect(fill = "white", colour = "white"),
    legend.box.spacing = unit(0.01, "line"),
    legend.key.height = unit(0.5, "lines"),
    legend.key.width = unit(0.5, "lines"),
    panel.border = element_rect(
      fill = "transparent",
      colour = "black",
      size = 0.2
    )
  ) +
  labs(
    colour = "Predicted feature",
    fill = "Parts"
  )

(plot_combined_tree_lengths <- plot_kappa_casein_tree +
  plot_indels_kappacasein_parts +
  plot_layout(widths = c(0.5, 2), ncol = 2)
)

save_plot(
  plot = plot_combined_tree_lengths,
  name = "figure_2"
)
```

# Figure 3

```{r figure3, fig.height=4.1, fig.width=5.95, cache=TRUE}
group_text_aa <- tribble(
  ~x, ~label,
  2.5, "polar non charged\n",
  7, "hydrophobic\n",
  11, "aromatic\n",
  14, "charged\n➕ ",
  16.5, "charged\n➖",
  19, "other\n",
)


(plot_aa_comp_range <- kappa_caseins_aa_comp_df_plot %>%
  ggplot(mapping = aes(
    x = as.numeric(residues)
  )) +
  geom_errorbar(mapping = aes(
    ymin = min,
    ymax = max,
    colour = part
  ), size = 0.8,
  position = position_dodge(width = 0.8)) +
  geom_vline(xintercept = c(4.5, 9.5, 12.5, 15.5, 17.5), colour = "darkgrey", size = 0.05) +
  geom_text(data = group_text_aa, mapping = aes(x = x, label = label), size = 3, y = Inf, vjust = 1.5, inherit.aes = F, lineheight = 0.8, color = "black") +
  geom_point(data = proteins_aa_comp_df_plot, mapping = aes(y = average, shape = dataset), colour = "black", show.legend = TRUE) +
  theme_minimal() +
  scale_x_continuous(breaks = 1:20, labels = list_residues_ordered_aa_comp, sec.axis = dup_axis(labels = function(x) {one_2_three_aas[list_residues_ordered_aa_comp[x]]}), expand = c(0.01, 0)) +
  scale_shape_manual(values = c("SwissProt" = 5)) +
  scale_colour_manual(values = c(colour_parts_kappa_casein), guide = guide_legend(override.aes = list(shape = NA))) +
  scale_y_continuous(expand = c(0.0, 0), sec.axis = dup_axis(), limits = c(NA, 0.34)) +
  labs(
    y = "frequency",
    x = "amino acid",
    colour = "Min/max frequencies",
    shape = "Average frequency"
  ) +
  theme(
    text = element_text(colour = "black", family = "Arial"),
    axis.text = element_text(colour = "black", family = "Arial"),
    axis.ticks = element_line(size = 0.1),
    axis.text.x.top = element_text(angle = 45, hjust = 0),
    axis.title.x = element_blank(),
    axis.title.y.right = element_blank(),
    panel.grid = element_blank(),
    panel.grid.major = element_blank(),
    panel.grid.major.x = element_line(size = 0.1, colour = "grey", linetype = "dotted"),
    panel.grid.minor.x = element_blank(),
    strip.text = element_text(hjust = 0, size = 10),
    panel.spacing.x = unit(1, "lines"),
    panel.border = element_rect(colour = "black", size = 0.9, fill = "transparent"),
    legend.position = "bottom",
    legend.justification = c(0, 1),
    legend.background = element_rect(colour = "white", fill = "white", size = NA),
    legend.direction = "horizontal",
    legend.box.margin = margin(0, 0, 0, 0),
    legend.margin = margin(0, 0, 0, 0),
    legend.box.spacing = unit(0.1, "lines"),
    plot.margin = margin(1, 1, 1, 1),
    legend.title = element_text(size = 9)
  ) +
  NULL
)

save_plot(
  plot = plot_aa_comp_range,
  name = "figure_3"
)
```

# Figure 4

```{r figure4, fig.height=8.5, fig.width=6, cache=TRUE}
# names and labels ----
label_other_residues <- "other"
label_glutamine <- "glutamine (Q)"
label_asparagine <- "asparagine (N)"
label_tyrosine <- "tyrosine (Y)"
label_other_aromatic <- "phenylalanine (F) or tryptophan (W)"
label_charged_minus <- "negatively charged (D or E)"
label_charged_plus <- "positively charged (R, K or H)"
label_pSer <- "phosphorylated serine (pS)"
label_Ser <- "Serine (S)"
label_Ch <- "goat" # Capra hircus
label_Bt <- "cow" # Bos taurus 



# other residues diplayed in grey ----
colour_other_residues <- c()
colour_other_residues[label_other_residues] <- "#e7e7e7"

# colours for each plot ----
colours_asngln <- colour_other_residues
colours_asngln[label_glutamine] <- "#002016"
colours_asngln[label_asparagine] <- "#009E73"

colours_aromatics <- colour_other_residues
colours_aromatics[label_other_aromatic] <- "#CC79A7"
colours_aromatics[label_tyrosine] <- "#461b32"

colours_charged <- colour_other_residues
colours_charged[label_charged_minus] <- "#e41a1c"
colours_charged[label_charged_plus] <- "#377eb8"


colours_phosphorylations <- colour_other_residues
colours_phosphorylations[label_pSer] <- "#247a62"
colours_phosphorylations[label_Ser] <- "#8da0cb"
colours_known_pSer <- c()
colours_known_pSer[label_Bt] <- "#a65628"
colours_known_pSer[label_Ch] <- "#4daf4a"

# vertical line at PKC/GMP cleavage site
# vline_cleavage <- geom_vline(
#   xintercept = position_align_chymosin_cleavage_site + 0.5,
#   colour = "black",
#   size = 0.2
# )


# weight residues according to species tree ----
fct_weight_conservation <- . %>%
  select(-seq_index, -residue) %>%
  left_join(kappa_caseins_phylo_tree_weights, by = "species") %>%
  group_by(msa_index, class) %>%
  summarise(
    n_w = sum(weight_norm)
  ) %>%
  ungroup()

# hydrophobicity legend ----
kd_range_max <- max(KyteDoolittle_scale_df$hydrophobicity)
kd_range_min <- min(KyteDoolittle_scale_df$hydrophobicity)
hydrophobicity_breaks <- c(kd_range_min, 2, 0, -2, kd_range_max)

# known phosphorylated serines ----
# from Uniprot
# goat: https://www.uniprot.org/uniprot/P02670#ptm_processing
# cow: https://www.uniprot.org/uniprot/P02668#ptm_processing
known_pSer_site_df <- tribble(
  ~msa_index, ~label, ~y,
  355, label_Ch, Inf,
  294, label_Bt, 0.91,
  294, label_Ch, Inf,
  179, label_Bt, 0.91,
  179, label_Ch, Inf
)

# Panel A - glutamine and asparagine ----

# dataset
df_plot_asngln <- kappa_caseins_alignment_positions %>%
  filter(residue != "-") %>%
  mutate(
    class = case_when(
      residue == "Q" ~ label_glutamine,
      residue == "N" ~ label_asparagine,
      TRUE ~ label_other_residues
    ) %>%
      fct_relevel(label_other_residues, label_asparagine, label_glutamine) # order colours for plot
  ) %>%
  fct_weight_conservation()

# plot
plot_alignment_asngln <- df_plot_asngln %>%
  ggplot(mapping = aes(y = n_w, x = msa_index, fill = class)) +
  geom_bar(stat = "identity", colour = NA) +
  scale_fill_manual(
    values = colours_asngln,
    guide = guide_legend(reverse = TRUE)
  ) +
  geom_rug(data = kappa_caseins_alignment_positions_parts, mapping = aes(x = msa_index, colour = part), outside = TRUE, sides = "b", inherit.aes = FALSE, show.legend = FALSE) +
  coord_cartesian(clip = "off") +
  scale_colour_manual(values = c(colour_parts_kappa_casein)) +
  labs(fill = "Residue") +
  NULL



# Panel B - tyrosines and aromatics ----

# dataset
df_plot_aromatics <- kappa_caseins_alignment_positions %>%
  filter(residue != "-") %>%
  mutate(
    class = case_when(
      residue == "Y" ~ label_tyrosine,
      residue %in% c("F", "W") ~ label_other_aromatic,
      TRUE ~ label_other_residues
    ) %>%
      fct_relevel(label_other_residues, label_other_aromatic, label_tyrosine) # order colours for plot
  ) %>%
  fct_weight_conservation()


# plot
plot_alignment_aromatics <- df_plot_aromatics %>%
  ggplot(mapping = aes(y = n_w, x = msa_index, fill = class)) +
  geom_bar(stat = "identity", colour = NA) +
  scale_fill_manual(
    values = colours_aromatics,
    guide = guide_legend(reverse = TRUE)
  ) +
    geom_rug(data = kappa_caseins_alignment_positions_parts, mapping = aes(x = msa_index, colour = part), outside = TRUE, sides = "b", inherit.aes = FALSE, show.legend = FALSE) +
  coord_cartesian(clip = "off") +
  scale_colour_manual(values = c(colour_parts_kappa_casein)) +
  labs(fill = "Residue") +
  NULL



# Panel C - charged residues ----

# dataset
df_plot_charged <- kappa_caseins_alignment_positions %>%
  filter(residue != "-") %>%
  mutate(
    class = case_when(
      residue %in% c("R", "K", "H") ~ label_charged_plus,
      residue %in% c("E", "D") ~ label_charged_minus,
      TRUE ~ label_other_residues
    ) %>%
      fct_relevel(label_other_residues, label_charged_plus, label_charged_minus) # order colours for plot
  ) %>%
  fct_weight_conservation()

# plot
plot_alignment_charged <- df_plot_charged %>%
  ggplot(mapping = aes(y = n_w, x = msa_index, fill = class)) +
  geom_bar(stat = "identity", colour = NA) +
  scale_fill_manual(
    values = colours_charged,
    guide = guide_legend(reverse = TRUE)
  ) +
    geom_rug(data = kappa_caseins_alignment_positions_parts, mapping = aes(x = msa_index, colour = part), outside = TRUE, sides = "b", inherit.aes = FALSE, show.legend = FALSE) +
  coord_cartesian(clip = "off") +
  scale_colour_manual(values = c(colour_parts_kappa_casein)) +
  labs(fill = "Residue") +
  NULL


# Panel D - predicted disorder ----

# dataset
df_plot_disorder <- kappa_casein_disorder_align %>%
  filter(!is.na(seq_index)) %>%
  mutate(
    class = cut(iupred_short, breaks = seq(0, 1, 0.2))
  ) %>%
  fct_weight_conservation()


# plot
plot_alignment_disorder <- df_plot_disorder %>%
  ggplot(mapping = aes(y = n_w, x = msa_index, fill = class)) +
  geom_bar(stat = "identity", colour = NA) +
  scale_fill_brewer(palette = "Greens") +
    geom_rug(data = kappa_caseins_alignment_positions_parts, mapping = aes(x = msa_index, colour = part), outside = TRUE, sides = "b", inherit.aes = FALSE, show.legend = FALSE) +
  coord_cartesian(clip = "off") +
  scale_colour_manual(values = c(colour_parts_kappa_casein)) +
  labs(fill = "Predicted disorder") +
  NULL


# Panel E - hydrophobicity ----

# dataset
df_plot_hydrophobicity <- kappa_caseins_hydrophobicity %>%
  filter(residue != "-") %>%
  rename(class = hydrophobicity) %>%
  fct_weight_conservation()

# plot
plot_alignment_hydrophobicity <- df_plot_hydrophobicity %>%
  ggplot(mapping = aes(y = n_w, x = msa_index, fill = class)) +
  geom_bar(stat = "identity", colour = NA) +
  scale_fill_distiller(
    palette = "BrBG",
    limits = c(kd_range_min, kd_range_max),
    direction = 1,
    breaks = sort(hydrophobicity_breaks),
    guide = guide_legend(barwidth = 10, barheight = 0.4, label.theme = element_text(size = 6))
  ) +
    geom_rug(data = kappa_caseins_alignment_positions_parts, mapping = aes(x = msa_index, colour = part), outside = TRUE, sides = "b", inherit.aes = FALSE, show.legend = FALSE) +
  coord_cartesian(clip = "off") +
  scale_colour_manual(values = c(colour_parts_kappa_casein)) +
  labs(fill = "Hydrophobicity") +
  NULL


# Panel F - phosphorylations ----

# dataset
df_plot_phosphorylations <- kappa_casein_fam20c_matches_tidy %>%
  mutate(match = TRUE) %>%
  rename(seq_index = match_position) %>%
  full_join(kappa_caseins_alignment_positions, by = c("species", "seq_index")) %>%
  mutate(match = !is.na(match)) %>%
  filter(residue != "-") %>%
  mutate(
    class = case_when(
      match == TRUE ~ label_pSer,
      residue == "S" ~ label_Ser,
      TRUE ~ label_other_residues
    ) %>%
      fct_relevel(label_other_residues, label_Ser, label_pSer) # order colours for plot
  ) %>%
  fct_weight_conservation()

# plot
plot_alignment_phosphorylations <- df_plot_phosphorylations %>%
  ggplot(mapping = aes(y = n_w, x = msa_index, fill = class)) +
  geom_bar(stat = "identity", colour = NA) +
  geom_text(data = known_pSer_site_df, mapping = aes(x = msa_index, colour = label, y = y), vjust = 1, label = "▼", inherit.aes = FALSE, size = 3) +
    geom_rug(data = kappa_caseins_alignment_positions_parts, mapping = aes(x = msa_index, colour = part), outside = TRUE, sides = "b", inherit.aes = FALSE, show.legend = FALSE) +
  coord_cartesian(clip = "off") +
  scale_colour_manual(values = c(colour_parts_kappa_casein, colours_known_pSer), breaks = c("cow", "goat")) +
  scale_fill_manual(
    values = colours_phosphorylations,
    guide = guide_legend(reverse = TRUE)
  ) +
  labs(
    fill = "Residue",
    colour = "Known phosphorylation sites"
  ) +
  guides(fill = guide_legend(order = 1, reverse = TRUE)) +
  NULL


# combine all plots ----

(plot_alignment_combined <- {
  (plot_alignment_asngln + labs(tag = "A")) /
    (plot_alignment_aromatics + labs(tag = "B")) /
    (plot_alignment_charged + labs(tag = "C")) /
    (plot_alignment_disorder + labs(tag = "D")) /
    (plot_alignment_hydrophobicity + labs(tag = "E")) /
    (plot_alignment_phosphorylations + labs(tag = "F"))
} + plot_layout(ncol = 1) &
  # vline_cleavage & # following scales, themes and labs applied to all subplots
  scale_x_align_residue_plot_pkc_gmp &
  scale_y_align_residue_plot &
  theme_bw() &
  theme(
    text = element_text(family = "Arial", colour = "black"),
    legend.position = "top",
    legend.title = element_text(size = 7),
    legend.text = element_text(size = 7),
    legend.box.spacing = unit(0.01, "line"),
    axis.text = element_text(size = 7, colour = "black"),
    axis.title = element_text(size = 7),
    panel.border = element_rect(size = 0.2),
    axis.ticks = element_line(size = 0.2),
    legend.key.width = unit(0.5, "lines"),
    legend.key.height = unit(0.5, "lines"),
    plot.tag.position = c(0, 1),
    plot.tag = element_text(colour = "black", size = 11),
    legend.justification = c(0, 0),
    legend.spacing.x = unit(0.4, "lines"),
    plot.margin = margin(0, 0, 0, 0),
    panel.grid.major.x = element_line(size = 0.01),
    panel.grid.minor.x = element_line(size = 0.01),
    panel.grid.major.y = element_line(size = 0.01),
    panel.grid.minor.y = element_line(size = 0.01),
  ) &
  labs(
    x = "aligned amino acid position",
    y = "weighted frequency"
  )
)

save_plot(
  plot = plot_alignment_combined,
  name = "figure_4"
)
```


# Figure 5

```{r figure5, fig.height=2.6, fig.width=6, cache=TRUE}
pH_breaks <- c(3, 4, 5, 6, 7)
pH_minor_breaks <- seq(1.5, 7.5, 0.5)
ncpr_highlighted_species <- c(
   "Squirrel monkey" = "Saimiri_boliviensis", # Black-capped ...
   "Guinea pig" = "Cavia_porcellus",
   "Human" = "Homo_sapiens",
   "House mouse" = "Mus_musculus",
   "Cattle" = "Bos_taurus"
)
geom_line_highlight_species <- partial(geom_line, mapping = aes(colour = species), size = 0.3)

# panel A
plot_ncpr_change_mature <- kappa_caseins_ncpr %>%
  filter(part == "mature") %>%
  ggplot(mapping = aes(x = pH, y = net_charge_per_residue, group = species)) +
  geom_hline(yintercept = 0, size = 0.1, linetype = "dotted") +
  geom_line(size = 0.1, colour = "lightgray") +
  facet_wrap(~part) +
  scale_y_continuous(
    limits = c(-0.21, 0.21)
  ) +
  scale_x_continuous(
    breaks = pH_breaks,
    minor_breaks = pH_minor_breaks,
    expand = c(0, 0)
  ) +
  scale_color_brewer(
    palette = "Set1",
    breaks = ncpr_highlighted_species,
    labels = glue("{names(ncpr_highlighted_species)}<br>_{underscore_to_space(ncpr_highlighted_species)}_"),
    guide = guide_legend(
      nrow = 1,
      ncol = 5,
      title.hjust = 0,
      label.hjust = 0,
      title.vjust = 1,
      label.vjust = 0,
      byrow = TRUE,
      direction = "horizontal"
    )
  ) +
  labs(
    y = "net charge per residue",
    x = "pH",
    colour = "Species"
  ) +
  theme_bw() +
  theme(
    legend.title = element_blank(),
    legend.position = "bottom",
    legend.justification = c(0, 1),
    panel.background = element_rect(size = 0.2, colour = "black"),
    panel.grid.major = element_line(size = 0.2),
    plot.tag.position = c(0, 1),
    strip.background = element_blank(),
    panel.spacing = unit(1.2, "line"),
    axis.text = element_text(size = 9, colour = "black"),
    axis.title = element_text(size = 10, colour = "black"),
    axis.ticks = element_line(size = 0.2, colour = "black"),
    legend.margin = margin(0, 0, 0, 200),
    legend.text.align = 0,
    legend.text = element_markdown(size = 8),
    legend.key.size = unit(10, "pt"),
    legend.box.spacing = unit(0.05, "line"),
  ) +
  NULL

# panel B
# same plot, different data
plot_ncpr_change_parts <- plot_ncpr_change_mature %+%
  (kappa_caseins_ncpr %>% filter(part != "mature")) &
  theme(
    legend.margin = margin(0, 0, 0, 0),
    legend.text.align = 1,
    legend.text = ggtext::element_markdown(size = 8),
    legend.key.size = unit(10, "pt"),
    legend.position = "none",
    legend.box.spacing = unit(0.05, "line"),
    legend.justification = c(1, 0)
    )

# combination of the 2 plots
# highlight species of interests
(plot_charge_combined <- (
  plot_ncpr_change_mature +
    geom_line_highlight_species(
      data = subset(
        kappa_caseins_ncpr,
        part == "mature" &
          species %in% ncpr_highlighted_species
      )
    ) +
    labs(tag = "A")
) +
  (
    plot_ncpr_change_parts +
      geom_line_highlight_species(
        data = subset(
          kappa_caseins_ncpr,
          part != "mature" &
            species %in% ncpr_highlighted_species
        )
      ) +
      labs(tag = "B")
  ) +
  plot_layout(widths = c(1, 2)) &
    theme(text = element_text(colour = "black", family = "Arial")) &
    coord_cartesian(clip = "off")
)


save_plot(
  plot = plot_charge_combined,
  name = "figure_5"
)
```


# Figure 6

```{r figure6, fig.height=7.5, fig.width=6, cache=TRUE}
# merge predicted count phosphorylations/o-glycosylations
df_phospho_glyco_counts <- bind_rows(
  phosphorylation = kappa_casein_fam20c_counts,
  Oglycosylation = kappa_casein_oglyc_glycomine_counts,
  .id = "PTM"
) %>%
  order_species_tree() %>%
  order_cleavage_parts() %>%
  mutate(n = if_else(part == "PKC", -n, n)) # uses negative values for PKC

# plot phosphorylations
plot_phospho_counts <- df_phospho_glyco_counts %>%
  filter(PTM == "phosphorylation") %>%
  ggplot(mapping = aes(x = n, y = species, fill = part)) +
  geom_barh(stat = "identity") +
  scale_fill_manual(values = c(colour_parts_kappa_casein)) +
  scale_y_discrete(expand = c(0.005, 0.0), position = NULL) +
  scale_x_continuous(
    expand = c(0.005, 0),
    limits = c(-8, 8),
    breaks = seq(-8, 8, 2),
    minor_breaks = seq(-8, 8, 1),
    labels = c(seq(8, 2, -2), 0, seq(2, 8, 2)),
    sec.axis = dup_axis()
  ) +
  theme_bw() +
  theme(
    axis.text.y = element_blank(),
    axis.ticks.y = element_blank(),
    legend.position = "bottom",
  ) +
  labs(
    title = "Phosphorylations",
    x = "count of predicted sites",
    fill = "Parts"
  ) +
  NULL

# plot glycosylations
plot_glyco_counts <- df_phospho_glyco_counts %>%
  filter(PTM == "Oglycosylation") %>%
  ggplot(mapping = aes(x = n, y = species, fill = part)) +
  geom_barh(stat = "identity") +
  scale_fill_manual(values = c(colour_parts_kappa_casein)) +
  scale_y_discrete(
    expand = c(0.005, 0.0),
    position = "right",
    labels = underscore_to_space
  ) +
  scale_x_continuous(
    expand = c(0.005, 0),
    limits = c(-20, 20),
    breaks = seq(-20, 20, 5),
    minor_breaks = seq(-20, 20, 1),
    labels = c(seq(20, 5, -5), 0, seq(5, 20, 5)),
    sec.axis = dup_axis()
  ) +
  theme_bw() +
  theme(
    legend.position = "none",
    axis.text.y = element_text(face = "italic", size = 6),
  ) +
  labs(
    title = "O-glycosylations",
    x = "count of predicted sites"
  ) +
  NULL

(plot_phospho_glyco_counts <- {
  plot_kappa_casein_tree + {
    {
      plot_phospho_counts + plot_glyco_counts
    } &
      theme(
        text = element_text(colour = "black", family = "Arial"),
        plot.margin = margin(0, 3, 0, 3),
        axis.title.y = element_blank(),
        axis.text.x = element_text(size = 7),
        axis.ticks = element_line(size = 0.1, lineend = "butt"),
        plot.title = element_text(size = 9),
        plot.subtitle = element_blank(),
        axis.title.x = element_text(size = 8),
        axis.title.x.top = element_blank(),
    legend.justification = c(0, 1),
    legend.title = element_text(size = 8),
    legend.text = element_text(size = 8),
    legend.background = element_rect(fill = "transparent"),
    panel.background = element_rect(fill = "white", colour = "white"),
    legend.box.spacing = unit(0.01, "line"),
    legend.key.height = unit(0.5, "lines"),
    legend.key.width = unit(0.5, "lines"),
        panel.grid.major.x = element_line(size = 0.1),
        panel.grid.major.y = element_line(size = 0.1),
        panel.grid.minor.x = element_blank()
      ) &
      geom_vline(xintercept = 0, size = 0.05, colour = "black")
  }
} +
  plot_layout(widths = c(0.7, 4)) 
)

save_plot(
  plot = plot_phospho_glyco_counts,
  name = "figure_6"
)
```



# Figure 7

```{r figure7, fig.height=8.25, fig.width=6, cache=TRUE}
all_cysteines_positions_rel_cattle <- kappa_caseins_cysteines_positions_rel_cattle %>%
  pull(cattle_position) %>%
  unique()
SS_bonds_categories <- kappa_caseins_SS_bonds_classes %>%
  pull(bond) %>%
  levels()

# kappa-casein residues for every position with a cysteine in a species
plot_kappa_casein_cysteines <- kappa_caseins_cysteines_positions_rel_cattle %>%
  mutate(position_id = group_indices(., cattle_position)) %>% # to simulate continuous axis
  ggplot(mapping = aes(
    x = position_id,
    y = species,
    label = residue,
    fill = group
  )) +
  geom_tile(mapping = aes(alpha = group)) +
  geom_text(family = "Source Code Pro", size = 1.9) +
  scale_fill_manual("Amino acids", values = aa_custom_OkabeIto_colours) +
  scale_alpha_manual("Amino acids", values = c("cysteine" = 1, set_names(rep(0.6, 19), c("histidine", "aromatic", "glycine", "proline", "polar", "apolar", "charged +", "charged -")))) +
  theme_bw() +
  theme(legend.position = "bottom") +
  scale_x_continuous(
    breaks = 1:12,
    labels = all_cysteines_positions_rel_cattle,
    expand = c(0, 0),
    sec.axis = dup_axis()
  ) +
  scale_y_discrete(
    expand = c(0, 0)
  ) +
  guides(fill = guide_legend(nrow = 9, ncol = 1, override.aes = list(alpha = 1))) +
  theme(
    text = element_text(colour = "black", family = "Arial"),
    axis.ticks.y = element_blank(),
    axis.text.y = element_blank(),
    legend.direction = "vertical",
    legend.position = c(-0.3, 1),
    legend.justification = c(0, 1),
    legend.key.width = unit(0.25, "lines"),
    legend.key.height = unit(0.25, "lines"),
    legend.title = element_text(size = 7),
    legend.text = element_text(size = 7),
    legend.box.margin = margin(2, 2, 2, 2, unit = "pt"),
    legend.margin = margin(2, 2, 2, 2, unit = "pt"),
    legend.background = element_rect(fill = "transparent"),
    legend.box.background = element_blank(),
    legend.spacing = unit(0.7, "mm"),
    axis.title.y = element_blank(),
    axis.title.x = element_text(size = 7),
    axis.text.x = element_text(size = 7),
    axis.ticks = element_line(size = 0.2),
    panel.border = element_rect(size = 0.05, colour = "black"),
    panel.grid.major.x = element_blank(),
    plot.margin = margin(0, 0, 0, 0),
    panel.grid.major.y = element_line(size = 0.01)
  ) +
  labs(
    x = "Bovine mature kappa-casein residue position",
    fill = "Amino acids",
    colour = "Amino acids"
  ) +
  NULL


# possibilities to form dimer/oligo/intrachain SS bonds
plot_SS_bonds <- kappa_caseins_SS_bonds_classes %>%
  filter(possibility) %>%
  arrange(bond) %>%
  mutate(bond_id = group_indices(., bond)) %>% # to simulate continuous axis
  ggplot(mapping = aes(x = bond_id, y = species)) +
  geom_text(label = "\u2713", size = 4) + # draw a tick
  geom_vline(xintercept = c(1.5, 2.5), size = 0.1, colour = "black") +
  scale_y_discrete(
    position = "right",
    labels = underscore_to_space,
    drop = FALSE
  ) +
  scale_x_continuous(
    breaks = 1:3,
    labels = SS_bonds_categories,
    sec.axis = dup_axis(),
    expand = c(0.25, 0)
  ) +
  theme_bw() +
  theme(
    legend.position = "none",
    axis.text.y = element_text(size = 5, face = "italic", colour = "black"),
    axis.title.x = element_text(size = 7),
    panel.border = element_rect(size = 0.1, colour = "black"),
    panel.grid.major.x = element_blank(),
    axis.title.y = element_blank(),
    axis.text.x = element_text(size = 7),
    axis.ticks = element_line(size = 0.2),
    plot.margin = margin(0, 0, 0, 0),
    panel.grid.major.y = element_line(size = 0.01)
  ) +
  labs(
    x = "predicted possible type of bonds"
  ) +
  NULL

(plot_combined_cysteines <- {
  plot_kappa_casein_tree + plot_kappa_casein_cysteines + plot_SS_bonds &
    theme(
      plot.margin = margin(0, 0, 0, 0),
      )
} +
  plot_layout(widths = c(0.5, 2, 0.9)) +
  NULL
)


save_plot(
  plot = plot_combined_cysteines,
  name = "figure_7"
)
```


# Figure 8

```{r figure8, fig.height=8.25, fig.width=6, cache=TRUE}
# data sets
pepsin_positions <- kappa_caseins_cleavage_positions %>%
  filter(peptidase == "pepsin") %>%
  left_join(kappa_caseins_alignment_positions, by = c("species", "cleavage_position" = "seq_index")) %>%
  order_species_tree()
plasmin_positions <- kappa_caseins_cleavage_positions %>%
  filter(peptidase == "plasmin") %>%
  left_join(kappa_caseins_alignment_positions, by = c("species", "cleavage_position" = "seq_index")) %>%
  order_species_tree()

plot_kappa_casein_pepsin <- plot_kappa_caseins_parts +
  geom_segment(
    size = 0.1,
    data = pepsin_positions,
    mapping = aes(
      x = msa_index + 0.5,
      xend = msa_index + 0.5,
      y = as.integer(species) - 0.5,
      yend = as.integer(species) + 0.5,
      colour = "predicted position"
    ),
    inherit.aes = FALSE
  ) +
  theme(
    axis.text.y = element_blank(),
    axis.ticks.y = element_blank(),
        legend.position = "bottom",
    legend.justification = c(0, 1),
    legend.title = element_text(size = 8),
    legend.text = element_text(size = 8),
    legend.background = element_rect(fill = "transparent"),
    panel.background = element_rect(fill = "white", colour = "white"),
    legend.box.spacing = unit(0.01, "line"),
    legend.key.height = unit(0.5, "lines"),
    legend.key.width = unit(0.5, "lines")
  ) +
  labs(
    title = "Pepsin",
    colour = "Cleavage site",
    fill = "Parts"
    ) +
  scale_colour_manual(values = c("predicted position" = "black")) +
  guides(fill = guide_legend(order = 1))

plot_kappa_casein_plasmin <- plot_kappa_caseins_parts +
  geom_segment(
    size = 0.1,
    data = plasmin_positions,
    mapping = aes(
      x = msa_index + 0.5,
      xend = msa_index + 0.5,
      y = as.integer(species) - 0.5,
      yend = as.integer(species) + 0.5
    ),
    inherit.aes = FALSE
  ) +
  theme(
    legend.position = "none",
    axis.text.y = element_text(colour = "black", size = 6)
  ) +
  labs(title = "Plasmin")


(cleavage_sites_position_plot <- {
  plot_kappa_casein_tree + {
    plot_kappa_casein_pepsin +
      plot_kappa_casein_plasmin &
      theme(
        text = element_text(colour = "black", family = "Arial"),
        plot.margin = margin(0, 7, 0, 0),
        legend.key.height = unit(0.9, "lines"),
        legend.key.width = unit(0.9, "lines"),
        )
  }
} +
  plot_layout(width = c(0.8, 4, 4))
)

save_plot(
  plot = cleavage_sites_position_plot,
  name = "figure_8"
)
```

# Figure S1


```{r figureS1, fig.height=8, fig.width=8, cache=TRUE}
# positions clades
clades_to_annotate <- tribble(
  ~node, ~clade,
  101, "monotremes",
  197, "marsupials",
  103, "placentals"
) %>%
  left_join(trimmed_species_tree_as_df, by = "node") %>%
  select(clade, branch, y)

plot_kappa_casein_tree_clades <- plot_kappa_casein_tree +
  geom_text(
    data = clades_to_annotate,
    mapping = aes(x = branch, y = y, label = clade),
    vjust = -0.3,
    size = 2.8,
    hjust = 0.5
  )

plot_msa <- kappa_caseins_alignment_positions %>%
  filter(residue != "-") %>%
  left_join(amino.acid.classifications %>%
    filter(classification == "custom: charge and aromatic"),
  by = c("residue" = "residue")
  ) %>%
  ggplot(mapping = aes(x = msa_index, y = species, fill = class)) +
  geom_tile(size = 0, alpha = 0.85) +
  scale_colour_manual(values = c("tandem repeat" = "black", colour_parts_kappa_casein)) +
  scale_fill_manual(values = aa_custom_OkabeIto_colours) +
  geom_rug(data = kappa_caseins_alignment_positions_parts, mapping = aes(x = msa_index, colour = part), outside = TRUE, sides = "b", inherit.aes = FALSE, length = unit(0.005, "npc")) +
  coord_cartesian(clip = "off") +
  # scale_x_continuous(
  #   expand = c(0, 0),
  #   breaks = align_breaks,
  #   labels = align_breaks_labels,
  #   minor_breaks = align_minor_breaks
  # ) +
  scale_x_align_residue_plot_pkc_gmp +
  scale_y_discrete(
    position = "right",
    expand = c(0.005, 0.0),
    labels = underscore_to_space
  ) +
  labs(
    x = "amino acid position in alignment"
  ) +
  theme_bw() +
  theme(
    text = element_text(colour = "black", family = "Arial"),
    legend.position = "none",
    axis.title.y.right = element_blank(),
    plot.margin = margin(0, 0, 0, 0),
    panel.spacing = margin(0, 0, 0, 0),
    axis.text.y = element_text(face = "italic", size = 7, colour = "black"),
    panel.grid.major.x = element_blank(),
    panel.grid.minor = element_blank()
  ) +
  NULL


(plot_tree_msa <- plot_kappa_casein_tree_clades +
  plot_msa +
  plot_layout(ncol = 2, nrow = 1, widths = c(0.2, 0.7)) +
  NULL
)


save_plot(
  plot = plot_tree_msa,
  name = "figure_S1",
  width = 8,
  height = 8
)
```

# Figure S2
```{r figureS2, echo=TRUE, fig.height=4.1, fig.width=6, cache=TRUE}
# same plot, different data
(plot_compare_evodist_divtime_nocorrect <- plot_compare_evodist_divtime %+%
  (compare_evodist_divtime %>%
    filter(corrected == FALSE)) &
  theme(
    text = element_text(colour = "black", family = "Arial"),
    plot.tag = element_blank())
 )

save_plot(
  plot = plot_compare_evodist_divtime_nocorrect,
  name = "figure_S2"
)
```

# Figure S3

```{r figureS3, fig.height=2.25, fig.width=6, cache=TRUE}
species_old_indels <- c(
  "Human" = "Homo_sapiens",
  "Cattle" = "Bos_taurus",
  "Short-beaked  echidna" = "Tachyglossus_aculeatus",
  "Platypus" = "Ornithorhynchus_anatinus",
  "Gray short-tailed opossum" =  "Monodelphis_domestica",
  "Common brushtail possum" = "Trichosurus_vulpecula"
)


# tree
tips_to_drop_old_indels <- trimmed_species_tree_as_df %>%
  filter(isTip, !label %in% species_old_indels) %>%
  pull(node)

tree_old_indels <- trimmed_species_tree %>%
  drop.tip(tips_to_drop_old_indels)

tree_old_indels_df <- tree_old_indels %>%
  ggplot2::fortify()

plot_tree_old_indels <- tree_old_indels_df %>%
  ggtree(size = 0.4) +
  geom_treescale(x = 50, y = 2)

# "barplot"
aa_old_indels_df <- kappa_caseins_alignment_positions %>%
  filter(species %in% species_old_indels) %>%
  arrange(msa_index) %>%
  mutate(is_gap = residue == "-") %>% # remove positions with gaps in **all** selected species
  group_by(msa_index) %>%
  mutate(all_gaps = all(is_gap)) %>%
  ungroup() %>%
  filter(!all_gaps, !is_gap) %>%
  mutate(new_index = group_indices(., msa_index)) %>%
  select(-residue, -all_gaps) %>% # order plot according to the new tree
  mutate_at("species", as.character) %>%
  left_join(tree_old_indels_df, by = c("species" = "label")) %>%
  mutate(species = fct_reorder(species, y)) %>%
  select(species, new_index)

aa_old_indels_df_max_length <- max(aa_old_indels_df$new_index)


plot_bars_old_indels <- aa_old_indels_df %>%
  ggplot(mapping = aes(x = new_index, y = species)) +
  geom_tile(colour = "black", fill = "black") +
  theme_bw() +
  theme(
    text = element_text(family = "Arial", colour = "black"),
    axis.ticks = element_line(size = 0.2, colour = "black"),
    axis.title.y = element_blank(),
    axis.line.y = element_blank(),
    axis.text.y.right = element_markdown(colour = "black"),
    axis.title.x = element_text(colour = "black"),
    axis.text.x = element_text(colour = "black"),
    plot.margin = margin(0, 0, 0, 0),
    panel.background = element_rect(fill = "white", colour = "white"),
    plot.background = element_rect(fill = "white", colour = "white"),
    legend.box.spacing = unit(0.01, "line"),
    legend.key.size = unit(5, "pt"),
    panel.border = element_blank(),
    strip.background = element_blank(),
    plot.subtitle = element_blank()
  ) +
  labs(x = "amino acid position in alignment") +
  scale_x_continuous(
    expand = c(0.02, 0),
    breaks = c(1, seq(50, 300, 50), aa_old_indels_df_max_length),
    minor_breaks = seq(0, 300, 25)
  ) +
  scale_y_discrete(
    position = "right", 
    breaks = species_old_indels,
    labels = glue("{names(species_old_indels)}<br>_{underscore_to_space(species_old_indels)}_")
    ) +
  NULL


(plot_tree_old_indels <- plot_tree_old_indels +
  plot_bars_old_indels +
  plot_layout(widths = c(0.25, 1))
)


save_plot(
  plot = plot_tree_old_indels,
  name = "figure_S3"
)
```

# Figure S4

```{r figureS4, fig.height=4.5, fig.width=6, cache=TRUE}
(aacomp_scatter_plot <- kappa_caseins_parts_aa_composition %>%
  mutate(residues = fct_relevel(residues, list_residues_ordered_aa_comp)) %>%
  ggplot(mapping = aes(x = GMP, y = PKC)) +
  geom_abline(slope = 1, color = "darkgrey", size = 0.1) +
  geom_point(alpha = 0.3, size = 0.3) +
  facet_wrap(~residues, ncol = 5) +
  theme_bw() +
  theme(
    text = element_text(colour = "black", family = "Arial"),
    strip.background = element_rect(fill = "white", colour = "black", size = 0),
    strip.text = element_text(size = 9, margin = margin(1, 1, 1, 1, unit = "pt")),
    panel.border = element_rect(size = 0.1),
    panel.spacing.x = unit(1, "lines"),
    axis.text = element_text(colour = "black"),
    axis.ticks = element_line(colour = "black", size = 0.1),
    panel.grid = element_blank(),
    panel.grid.major = element_line(
      size = 0.1,
      linetype = "dotted",
      colour = "black"
    ),
    panel.grid.minor = element_line(
      size = 0.05,
      linetype = "dotted",
      colour = "darkgrey"
    ),
  ) +
  scale_x_continuous(
    limits = c(0, 0.31),
    expand = c(0, 0),
    breaks = seq(0, 0.4, 0.1)
  ) +
  scale_y_continuous(
    limits = c(0, 0.31),
    expand = c(0, 0), breaks = seq(0, 0.4, 0.1)
  ) +
  coord_equal() +
  labs(
    x = "amino acid frequency (GMP)",
    y = "amino acid frequency (PKC)"
  ) +
  NULL
)

save_plot(
  plot = aacomp_scatter_plot,
  name = "figure_S4"
)
```



# Figure S5

```{r figureS5, fig.height=8, fig.width=6, cache=TRUE}
# phosphoryaltion predictions
fam20c_dataset <- kappa_casein_fam20c_matches_tidy %>%
  left_join(kappa_caseins_alignment_positions, by = c("species", "match_position" = "seq_index")) %>%
  filter(!is.na(match_position))
# order_species_tree()

plot_kappa_casein_pred_phospho_positions <- plot_kappa_caseins_parts +
  geom_segment(
    size = 0.2,
    data = fam20c_dataset,
    mapping = aes(
      x = msa_index,
      xend = msa_index,
      y = as.integer(species) - 0.5,
      yend = as.integer(species) + 0.5,
      colour = "predicted position"
    ),
    inherit.aes = FALSE
  ) +
  theme(
    axis.text.y = element_blank(),
    axis.ticks.y = element_blank(),
    legend.position = "bottom",
    legend.justification = c(0, 1),
    legend.title = element_text(size = 8),
    legend.text = element_text(size = 8),
    legend.background = element_rect(fill = "transparent"),
    panel.background = element_rect(fill = "white", colour = "white"),
    legend.box.spacing = unit(0.01, "line"),
    legend.key.height = unit(0.5, "lines"),
    legend.key.width = unit(0.5, "lines")
  ) +
  labs(
    title = "Phosphorylations",
    colour = "PTM",
    fill = "Parts"
    ) +
  scale_colour_manual(values = c("predicted position" = "black")) +
  NULL

# O-glycosylation predictions
oglyc_dataset <- kappa_casein_oglyc_predictions %>%
  filter(Oglyc_pred) %>%
  left_join(kappa_caseins_alignment_positions, by = c("species", "position" = "seq_index")) %>%
  order_species_tree()

plot_kappa_casein_pred_oglyc_positions <- plot_kappa_caseins_parts +
  geom_segment(
    size = 0.2,
    data = oglyc_dataset,
    mapping = aes(
      x = msa_index,
      xend = msa_index,
      y = as.integer(species) - 0.5,
      yend = as.integer(species) + 0.5
    ),
    inherit.aes = FALSE
  ) +
  theme(
    axis.text.y = element_text(colour = "black", family = "Arial"),
    legend.position = "none"
  ) +
  labs(title = "O-glycosylations") +
  NULL

(plot_phospho_oglyco_positions <- plot_kappa_casein_tree + {
  plot_kappa_casein_pred_phospho_positions +
    plot_kappa_casein_pred_oglyc_positions &
    theme(
      text = element_text(colour = "black", family = "Arial"),
      plot.margin = margin(0, 8, 0, 0)
      )
} +
  plot_layout(widths = c(0.8, 4, 4))
)

save_plot(
  plot = plot_phospho_oglyco_positions,
  name = "figure_S5"
)
```

# Figure S6

```{r figure S6, fig.height=10, fig.width=4.44, cache=TRUE}
cysteines_positions <- kappa_caseins_alignment_positions %>%
  filter(residue == "C") %>%
  select(-seq_index, -residue)

plot_kappa_casein_cysteines <- plot_kappa_caseins_parts +
  geom_segment(
    size = 0.2,
    data = cysteines_positions,
    mapping = aes(
      x = msa_index,
      xend = msa_index,
      y = as.integer(species) - 0.5,
      yend = as.integer(species) + 0.5,
      colour = "position"
    ),
    inherit.aes = FALSE
  ) +
  scale_colour_manual(values = c("position" = "black")) +
  theme(
        legend.position = "bottom",
    legend.justification = c(0, 1),
    legend.title = element_text(size = 8),
    legend.text = element_text(size = 8),
    legend.background = element_rect(fill = "transparent"),
    panel.background = element_rect(fill = "white", colour = "white"),
    legend.box.spacing = unit(0.01, "line"),
    legend.key.height = unit(0.5, "lines"),
    legend.key.width = unit(0.5, "lines"),
    axis.text.y = element_text(colour = "black", family = "Arial"),
  ) +
  labs(colour = "Cysteines", fill = "Parts", title = "Cysteines") +
  NULL

(cysteines_position_plot <- {
  plot_kappa_casein_tree +
    plot_kappa_casein_cysteines
} +
  plot_layout(width = c(1, 4)) &
  theme(
    text = element_text(colour = "black", family = "Arial"),
    plot.margin = margin(0, 0, 0, 0)
    )
)

save_plot(
  plot = cysteines_position_plot,
  name = "figure_S6"
)
```

# Table S1

```{r tableS1, results='asis'}
# formating for html report
kappa_casein_gi %>%
  mutate_at("species", fct_relabel, underscore_to_space) %>%
  kable(format = "html") %>%
  kable_styling()


# formatting for latex and export
kappa_casein_gi %>%
  mutate_at("species", ~ glue("\\textit{<<.x>>}", .open = "<<", .close = ">>")) %>%
  mutate_at("species", ~ fct_recode(.x,
    "\\textit{Ictidomys_tridecemlineatus} \\textsuperscript{b}" = "\\textit{Ictidomys_tridecemlineatus}",
    "\\textit{Fukomys_damarensis} \\textsuperscript{c}" = "\\textit{Fukomys_damarensis}",
    "\\textit{Bos_mutus} \\textsuperscript{h}" = "\\textit{Bos_mutus}",
    "\\textit{Carlito_syrichta} \\textsuperscript{e}" = "\\textit{Carlito_syrichta}",
    "\\textit{Vicugna_pacos} \\textsuperscript{g}" = "\\textit{Vicugna_pacos}",
    "\\textit{Neomonachus_schauinslandi} \\textsuperscript{f}" = "\\textit{Neomonachus_schauinslandi}",
    "\\textit{Nannospalax_galili} \\textsuperscript{d}" = "\\textit{Nannospalax_galili}"
  )) %>%
  mutate_at("species", fct_relabel, underscore_to_space) %>%
  mutate_at("gi", ~ ifelse(is.na(.x), "\\textsuperscript{a}", .x)) %>%
  rename(
    `NCBI Taxon ID` = taxid,
    `NCBI GI Identifier` = gi
  ) %>%
  knitr::kable(
    format = "latex",
    booktabs = TRUE,
    linesep = "",
    longtable = TRUE,
    caption.short = "Kappa-casein GenInfo Identifiers",
    label = "gi_species",
    caption = "\\textbf{Kappa-casein GenInfo Identifiers.}",
    escape = F
  ) %>%
  kable_styling(
    latex_options = c(
      "striped",
      "hold_position",
      "full_width",
      "condensed",
      "repeat_header"
    ),
    full_width = TRUE,
    repeat_header_continued = TRUE
  ) %>%
  # group_rows("Monotremes", 1, 2) %>%
  # group_rows("Marsupials", 3, 4) %>%
  # group_rows("Placentals", 5, 99) %>%
  footnote(
    alphabet = c(
      "First exon was recovered from a BLAST with the elephant sequence", # a - Manatee
      "Was ``Spermophilus tridecemlineatus'' in @cite{fritz_2009}", # b - Ictidomys_tridecemlineatus
      "Was ``Cryptomys damarensis'' in @cite{fritz_2009}", # c - Fukomys_damarensis
      "Used instead of ``Spalax ehrenbergi'' in @cite{fritz_2009}", # d - Nannospalax galili
      "Was ``Tarsius syrichta'' in @cite{fritz_2009}''", # e - Carlito_syrichta
      "Was ``Monachus schauinslandi\" in @cite{fritz_2009}''", # f - Neomonachus schauinslandi
      "Was ``Vicugna vicugna'' in @cite{fritz_2009}", # g - Vicugna_pacos
      "Was ``Bos grunniens'' in @cite{fritz_2009}" # h - Bos mutus
    ),
    escape = FALSE
  ) %>%
  str_replace_all("@cite", "\\\\textcite") %>%
  save_table(table = ., name = "table_S1")
```

# Table S2

```{r tableS2, results='asis'}
# formating for html report
kappa.casein.manual.repeats %>%
  mutate_at("species", underscore_to_space) %>%
  kable(format = "html") %>%
  kable_styling()

format_species_ptr_table <- . %>%
  str_replace("Dipodomys_ordii", glue("Dipodomys_ordii \\{footnote_marker_symbol(1, 'latex')}")) %>%
  fct_relabel(underscore_to_space) %>%
  map_chr(~ glue("\\textit{<<.x>>}", .open = "<<", .close = ">>")) # italicized

format_sequence_ptr_table <- . %>%
  map_chr(~ str_remove_all(.x, "[ -]")) %>% # remove gaps
  map_chr(~ glue("\\texttt{<<.x>>}", .open = "<<", .close = ">>")) # monospaced

# formatting for latex and export
kappa.casein.manual.repeats %>%
  order_species_tree() %>%
  mutate_at("species", format_species_ptr_table) %>%
  mutate_at("sequence", format_sequence_ptr_table) %>%
  rename(N = N_flank_position, C = C_flank_position) %>%
  knitr::kable(
    format = "latex",
    booktabs = TRUE,
    linesep = "",
    longtable = TRUE,
    caption.short = "Protein tandem repeats found in kappa-casein sequences.",
    label = "supp_ptr",
    caption = "\\textbf{Protein tandem repeats found in kappa-casein sequences.} 
    Positions are given for the extremities of each tandem repeat unit 
    in the mature sequence.",
    escape = FALSE
  ) %>%
  add_header_above(c(" ", "position" = 2, " ")) %>%
  kable_styling(
    latex_options = c(
      "striped",
      "hold_position",
      "full_width",
      "condensed"
    ),
    full_width = TRUE,
    repeat_header_continued = TRUE
  ) %>%
  footnote(symbol = c("likely highly degenerate repeat")) %>% 
  column_spec(1, width = "1.5in") %>%
  column_spec(2, width = "0.5in") %>%
  column_spec(3, width = "0.5in") %>%
  save_table(table = ., name = "table_S2")
```

# Table S3

```{r tableS3, results='asis', fig.cap="test"}
# formating for html report
all_predictions_metrics %>%
  mutate_if(is.numeric, round, digits = 2) %>%
  kable(format = "html") %>%
  kable_styling()


# formatting for latex and export
all_predictions_metrics %>%
  mutate_if(is.numeric, round, digits = 2) %>%
  mutate(method = fct_recode(method,
    "GlycoMine" = "glycomine",
    "O-GlcNAcPRED-II" = "O-GlcNAcPRED-II",
    "NetOClyc4.0" = "netoglyc",
    "GlycoPred" = "glycopred"
  )) %>%
  kable(
    format = "latex",
    longtable = TRUE,
    booktabs = TRUE,
    caption.short = "Prediction of O-glycosylation in kappa-casein",
    label = "supp_glyco_pred_methods",
    caption = "\\textbf{Prediction of O-glycosylation in kappa-casein 
    with GlycoMine \\autocite{li_2015a}; 
    GlycoPred \\autocite{hamby_2008}; 
    O-GlcNAcPRED-II \\autocite{jia_2018}; 
    and NetOGlyc4.0 \\autocite{steentoft_2013}.}",
    escape = F
  ) %>%
  kable_styling(latex_options = c("striped", "hold_position", "full_width")) %>%
  save_table(table = ., name = "table_S3")
```

# Session info

```{r sessioninfo, echo=FALSE}
options(width = 95)
si <- sessioninfo::session_info()
writeLines(as.character(si), "session_info.txt")
si
```