Input Line 2: Secondary Structures

Sec.Str-File

Name of the file containing secondary structure information. By default, displayed secondary elements are extracted from the first monomer, but you can select a chainID with the command 'chain_': file1.2st chain_A

Remark: Three kinds of layouts are used, depending if one or two secondary structure files are supplied:

1. If one sec. structure file is supplied (uploaded in the box named top in webESPript)

Sec. str. elements are displayed at the top of each block of sequences and relative accessibility at the bottom.

2. If two sec. structure files are supplied:

Sec. str. elements of first file and corresponding accessibility are displayed at the top of each block

Sec. str. elements of second file (uploaded in the box named bottom in webESPript) and corresponding accessibility are displayed at the bottom of each block

3a. If file1.2st is entered as usual and the string none is entered as file2.2st: sec. str. elements and relative accessibility are displayed at the top of each block - see example1

3b. If the string none is entered as file1.2st and file2.2st is entered in turn: sec. str. elements of second file and relative accessibility are displayed at the bottom of each block.

By default, file1.2st (top in webESPript) and file2.2st (bottom in webESPript) refer to the first and the last displayed sequences. This default can be changed by using the Special Character X for the first sec. structure file and Z for the second.

Remark: sec. str. elements can be extracted by reading the alignment file file.aln, if you type the character * instead of file1.2st (click on 'extract info' in webESPript). This * option prevents you from typing file.aln twice and can be used for alignment files from Predict Protein or from NPS@, which contain information on predicted sec. structure elements - see example1.
You can also click on 'extract info' in webESPript to extract secondary structure information with DSSP, if you have uploaded a PDB file in the box 'aligned sequences'.

Acc-Disp [default: none]

Displays relative accessibility if you upload DSSP or PHD files as file1.2st or file2.2st.

ScoreConfidence [default: 9]

When the sec. structure file is a PHD file, secondary elements with a reliability equal at least to ScoreConfidence are highlighted. If reliability is below limit, helices appear as small squiggles, beta strands as dotted lines and labels are not written - see example1

AutomaticSearch [default: none]

ESPript searches in the directory $DSSP_DIR for files having the same name as aligned sequences. Thus, secondary structure information of each aligned sequence with a known 3D structure can be displayed.
This option implies that you have a database of DSSP files installed on your disk. It can be used with ENDscript, when you search for homologous sequences against the PDB.

Input Line 3: Output

Output-file

name of the PostScript output file.

NumberingOption

By default, alpha helices, 310 helices, 5-helices (pi) and beta strands are numbered with digits.

With the L option, helices and beta strands are numbered with letters, starting at 'A'. With the M option, helices are numbered with digits and strands with letters.

Remark: You can remove all sec. structure labels by using the Special Character command: A S all (button 'hide labels' in webESPript) if you want to prepare figures such example5.

SequenceOutput

The SEQ option allows to extract a single sequence in a one-letter code, from a multiple alignment file entered as file.aln. By default, this sequence correponds to the first displayed in the ESPript figure and is written in a file named file.seq. The extracted sequence can be used in NPS@ or other servers to perform further queries. The SEQ option can also be used to extract sequence information from a PDB file.

BobScriptOutput

The BOB option allows to generate a BOBSCRIPT command file, which is used in turn to generate a PostScript figure, showing secondary structure elements defined by DSSP or STRIDE and coloured according to sequence similarity (see appendix2). You can use ENDscript to obtain such a figure without too much pain.

Remark: You can use the command BOBL instead of BOB, in order to show in the PostScript file, side chains of residues strictly conserved.

Input Line 4: Similarity Score

Check appendix for a general view on similarity computation and colour scheme.

SimilarityGlobalScore [default: 0.7]

If R, B, P or I as SimilarityType: a global score is calculated for all sequences by extracting all possible pairs of residues per columns. If applicable, a second score is calculated within each group of sequences.

If S, M or E as SimilarityType: a percentage is calculated for each column of residues.

If the score is greater than SimilarityGlobalScore, it will be rendered as coloured characters (red characters on a white background by default and white characters on a red background if residues are strictly conserved in the column) with frames (blue by default). Note that strictly conserved residues are boxed but are not framed, if you enter a SimilarityGlobalScore greater than 1.

SimilarityDiffScore [default: 0.5]

Applicable if R, B, P or I as SimilarityType: residues which are conserved within a group but not conserved from one group to the other are highlighted (yellow background by default).

SimilarityType [default: R]

If R, B, P or I: a matrix is used to calculate the similarity score. Risler, Blosum62, Pam250 and Identity are the four possibilities. We recommend a SimilarityGlobalScore of 0.1-0.2 with B or P matrices and of 0.6-0.7 with R or I matrices.

If S: a percentage of Strictly conserved residues is calculated per columns.

If M: a percentage of similarity is calculated considering criteria used in MULTALIN.

IV ; LM ; FY ; NDQEBZ

HKR are polar positive ; DE are polar negative ; STNQ are polar neutral ;

AVLIM are non polar aliphatic ; FYW are non polar aromatic ; PG ; C

Input Line 5: Output Layout

FontSize [default: 7]

Size in points for the fonts (Courier for sequence names and residues).

ColumnNb [default: 70]

Number of residue columns per line.

Remark: a column numbers value is calculated at the end of the log file (end of the OUT file in webESPript). This value can be re-entered in order to obtain a justified figure (look for the sentence 'suggestion columns per line' in the log file or in OUT).

Vgap [default: 6]

Vertical gap between two blocks of sequences. The unit for the distance is the height of a line.

Vshift [default: 0]

Vertical shift for the whole display. The unit for the distance is the height of a line.

Hshift [default: 0, centred]

Horizontal shift for the whole display. The unit for the distance is the width of a residue.

Bshift [default: 0]

Shift lines below bottom sequence. The unit for the distance is the width of a residue.

PrinterOpt [default: C]

C coloured output ; T coloured with all letters in bold, ideal for thermal printers or any others, before reduction of your figure in an article ; S light yellow background, ideal for slides ; B black & white, a grey scale is used (see standard colours in section Special Characters) ; F flashy colours, similar residues are written with black bold characters and boxed in yellow, ideal for overheads.

Paper [default: P]

P: Portrait(A4) ; L: Landscape(A4) ; P3: Portrait(A3) ; L3: Landscape(A3).

AllNumbered [default: first sequence]

By default, the first sequence is numbered every ten residues as in example2. With the option N (click on 'number sequences' in webESPript) all sequences are numbered at the beginning of each block of sequences as in example3

Input Lines 6: Special Commands and Characters

hide sequences

Aligned sequences are not written (click on 'hide sequence' in webESPript). '@skip' is a shortcut for the block of Special Characters below:
I S all ! skip all
F S all ! "
H S all ! "
B S all ! "
O S all ! "
N S all ! "
T S all ! "
Y S all ! "
This option can be used to build a figure including several secondary structure elements as in example4.

more info from a file from Predict Protein or from NPS@

Extra information can be extracted upon use of the command '@pp', if
1. a mail from the Predict Protein server is entered as file.aln.

- ProDom domains are visualized with yellow bars below each block of sequences

- x marks from the SEG low-complexity¹ search are represented with dotted lines

- peptides resulting from a PROSITE² search are shown with bold letters

2. a file from the NPS@ server with multiple sequence alignment and predicted secondary structure elements is entered as file.aln.

- predicted sec.structure elements are shown below each aligned sequence: i.e. helices with squiggles, beta strands with arrows, ambiguous predictions with solid circles

minus or plus

Residue numbering can be changed along a single sequence. If '@minus' is used, numbering is shifted by -1 at the given column (here at columns 5 and 40). If '@plus' is used, residue numbering is shifted by +1 at the given column. Before using this option, use the command '@ruler' described below to visualize column numbers.

Remark: '@minus' and '@plus' are equivalent to the buttons 'delete in seq' and 'insert in seq' in webESPript
Note that by default sequence numbering refers to the first displayed sequence, but it can refer to the third displayed sequence (for example) if you enter the Special Command Y D 3.

preview column numbers

Column numbers are displayed on the PostScript image. This option is useful when preparing a figure with the special commands '@minus' or '@plus' presented above, or the Special Characters Q, V, W (section Do_it).

insert text at sequences

The command is: @seq [sequence number or sequence name] [text or blank]
The text is then inserted above the chosen sequence. Note that sequences numbers are given in the log file of ESPript.

Special case: the text is inserted below the last displayed sequence, if you chose a number greater than the number of displayed sequences. Thus, you can give a name to a line of Special Characters and change the colour of the name with the Special Character T. As a test, you can enter in example2 the two lines of command below:
@seq 100 important residues
T R 100

modify or create colours

Assigns a new rgb code for a Special Characters colour in ESPript (here red, R, and blue, B, colours are modified).
You can also create a new special character colour, such as A for grey:
@col A .5 .5 .5 ! create a new colour named A
I A all      ! strictly conserved residues are in grey

Remark: a new character colour must be created before being used as in the example above. S is reserved to skip. Otherwise, any uppercase character can be used. Have a look at this site to chose new colours and corresponding percent rgb values (range is 0-1 and white is 1 1 1).

replace labels

Sec.structure labels can be replaced by new ones defined by the user. Labels starting by a, b, h, p refer to alpha helices, beta strands, 310 helices and 5-helices respectively. These first characters are not displayed. Replacement is made according to the order of entrance (see example4), firstly through the top sec. structure elements, then through the bottom sec. structure elements if applicable.
Command lines can be written with all alpha helices firstly, then all beta strands, 310 and 5-helices (for instance you can remove labels of all 310 helices by typing as many @h h h h h as needed).

Remark: If the first letter is typed in uppercase (@Ag1 Ag2), the second letter is displayed using a Symbol font (here, displayed labels would be gamma1 gamma2).

hide turns

Strict alpha and beta turns, usually rendered as TTT and TT, are not displayed (see information on secondary structures).

insert secondary structure elements

Inserts alpha helices (a), beta strands (b), 310 helices (h) or 5-helices (p) at the top or bottom of sequences blocks. Rules of numbering are the same as in section Secondary Structures (i.e. by default, top and bottom secondary structure elements match top and bottom sequences respectively).

Remark: You can enter up to 264 characters on this line of command. Click on the button +1 of the interface to duplicate the form, if you use webESPript and if you exceed this limit. Thus, you may be able to enter alpha helices in part [01], and beta strands in part [0 1], while still being under the limit of 264 characters in each part.

hide names of secondary structure elements

Removes the name of the corresponding sequence at the beginning of each line of sec. str. elements By default this name has the same colour as the first displayed element.

Remark: Assume a very special case, where your sequence starts at 10, and you want to colour sec.structure name in red and sec.structure elements in blue. Then you can use the Special Characters command X:
X R 10-10
X B 11-4500.

hide alternate conformations

hide disulphide bridges

substitute sequence names

Remark: Suppose you want to change the names of the first and third displayed sequences.
You can also type: @sub 1 newname1 3 newname2

Input Lines 6b: Special Characters

Character-Type	Character-Colour	Position
P, T, R, X, Y, Z, Q, V, W, U, D, G, J, S, C, E, L, K, A, I, F, M, H, B, O, N, s, t, u, a, b, c, d, e, f, g, h, i, j, k, l, m, n	D, B, R, P, G, C, O, Y, W, S	2 9-39
Example	U R 2 9-39
Mode in www	advanced

Entry on each line is Character-Type Colour Position. Thus, the command to display red triangles on residues 2 and from 9 to 39 is:
U R 2 9-39
By default, residues are numbered according to the first displayed sequence.

Character-Type

Type 1: miscellaneous

P calculates hydropathy ; T changes colour of sequence names ; R reads intermolecular contacts

Type 2: assignment

X top secondary structure information is assigned to a chosen sequence, which is the first one by default; colour of secondary elements can be changed ; Y sequence numbering is assigned to a chosen sequence, which is the first one by default; colour of digits can be changed

; Z residue numbering of another sequence, which is the last one by default, can be displayed at the bottom of sequences blocks ; secondary structure information corresponding to this sequence can also be displayed (see example3 )

Type 3: do it yourself

Q boxes residues (check example5) ; V bold characters ; W adds frames

Type 4a: adding markers

U triangle up (check example2); D triangle down ; G go ; J jammed ; S star ; C solid circle ; E open circle ; L dotted line

; K stroke

Type 4b: changing default colours of

A labels above top sec. structure elements ; I identity boxes ; F identity characters ; M group similarity boxes ; H group similarity characters ; B global similarity frames ; O difference similarity boxes ; N low similarity scores

Type 4c: adding NMR markers

s: Amide proton slow exchange rate (< 1mn^-1)

t: ³J_HN,HaNH-Ha coupling constant less than 6 Hz

u: ³J_HN,HaNH-Ha coupling constant > or equal to 7 Hz

a, b, c: d_NN(i,i+1) NOE between proton NH of residue i and i+1 (weak medium strong)

d, e, f: d_aN(i,i+1) NOE between proton alpha of residue i and proton NH of i+1 (weak medium strong)

g, h, i: d_bN(i,i+1) : NOE between proton beta of residue i and proton NH of i+1 (weak medium, strong)

j: d_NN(i,i+2) NOE between proton NH of residue i and proton NH of i+2

k: d_aN(i,i+2) : NOE between proton alpha of residue i and proton NH of i+2

l: d_aN(i,i+3) : NOE between proton alpha of residue i and proton NH of i+3

m: d_ab(i,i+3) : NOE between proton alpha of residue i and proton beta of i+3

n: d_aN(i,i+4) : NOE between proton alpha of residue i and proton NH of i+4

Character-Colour (except if R is Character-Type)

Dark(black), Blue, Red, Pink, Green, Cyan, Orange, Yellow, White, Skip(transparent)
Remark: a grey scale is used in mode black & white

Position, four cases, [] means mandatory and {} optional

1a. if P or T: [Character-Colour] [sequence name number or range, 1-1000 stands for all] {new sequence name number or range} {....}

Example to calculate hydropathy of the third displayed sequence: P R 3

(the string 'hyd' will be written in red) or to colour the name of the second sequence in green: T G 2
1b. if R: [ChainId] [residue range] {new residue range} {...}
checks appendix for details on intermolecular contacts

2. if X, Y or Z: [Character-Colour] [name or number of sequence displayed] {Start-Index, 1 by default} OR [residue range] {new residue range} {...}

Example to assign the first sec. str. file to the third displayed sequence: X B 3 (sec. structure elements are in blue), to number the fourth displayed sequence in blue: Z B 4 (the same command Z B 4 can be used to assign the second sec. structure file to the fourth displayed sequence and to colour sec. structure elements in blue).
You can also colour elements in blue and red as in the example below
X B 3 ! sec. str. elements refer to the 3 displayed sequence and are in blue. This sequence is now the reference
X R 4-50 60-80 ! but sec. structure elements from residues 4 to 50 and from 60 to 80 are in red

Remark: you can type X B name_of_the_third_displayed_sequence instead of X B 3 as in the example above.

3. if Q, V or W: [Character-Colour] [number or range of sequence displayed] {column range} {new column range} [...]

Note that column numbering is used in this case instead of residue numbering.
Use the special command @ruler to preview column numbers.
Example to highlight in yellow residues of sequences 3-8 from columns 40 to 45 and from 50 to 55: Q Y 3-8 40-45 50-55, or to highlight the last sequence in cyan: Q C 1000.

4. if U, D, S, C, L, A, I, F, M, H, B, O, N, s, t, u, a, b, c, d, e, f, g, h, i, j, k, l, m or n: [Character-Colour] [residue number or range] {new residue number or range} {...}

Example to add red triangles at residue 2 and from 9 to 39: U R 2 9-39, to box all identical residues in blue: I B 1-4500, to remove all sec. structure labels: A S 1-4500

By default, positions refer to residue numbering of the first displayed sequence. Use the special command Y to change this default.

Y B 3 ! residue numbering refers to the 3 displayed sequence and residues numbering is in blue
U R 9 20-30 ! adds red triangles below columns containing residues 9 and 20 to 30 of sequence 3

Remark: Enter one special character per line. You can repeat a residue range or a column range on this line. You can use the string all instead of 1-1000 in case 1a and of 1-4500 in cases 1b, 2, 3 and 4 (i.e. A S all to remove all secondary structure labels).

Input Line 6c: Comment

A line beginning with % will be displayed at the bottom of the generated PostScript, as a comment or a title.

Input Line 6d: Ending the section

A single dot on a line ends this section.

Input Lines 7...: Defining Groups and Blocks

You can select the sequences to be displayed and their order on a single line: 2 1 3-5
all can be used to select the rest of the sequences: 2 all (see example5)
A % before a sequence number keeps a sequence for similarity calculations but prevents it from displaying: 2 %1 %3-5 (see example4).

You can also separate your sequences in groups for similarity computations, each line defining a group and giving the order of the sequences to display as in example2 (ADVanced or EXPert modes in webESPript). The calculation by group is not performed if SimilarityType is Strict, Multalin or Equivalent (groups are just numbered).
This section is ended by a single dot on a single line.

Appendix

file.aln

• MULTALIN³ or PRODOM⁴
• CLUSTALW⁵
• NPS@-MPSA⁶
• GCG-MSF⁷
• FASTA⁸
• SEAVIEW-MASE⁹
• MAXHOM¹⁰ (enter the full mail from the Predict Protein server)
• THREADER¹¹
• PDB¹²

vp7_btv1s       MDTIAARALTVMRACATLQEARIVLEANVMEILGIAINRYNGLTLRGVTMRPTSLAQRNE
vp7_btv10       MDTIAARALTVMRACATLQEARIVLEANVMEILGIAINRYNGLTLRGVTMRPTSLAQRNE

vp7_btv1s       MFFMCLDMMLSAAGINVGPISPDYTQHMATIGVLATPEIPFTTEAANEIARVTGETSTWG
vp7_btv10       MFFMCLDMMLSAAGINVGPISPDYTQHMATIGVLATPEIPFTTEAANEIARVTGETSTWG

> vp7_btv1s
MDTIAARALTVMRACATLQEARIVLEANVMEILGIAINRYNGLTLRGVTMRPTSLAQRNE
MFFMCLDMMLSAAGINVGPISPDYTQHMATIGVLATPEIPFTTEAANEIARVTGETSTWG
> vp7_btv10
MDTIAARALTVMRACATLQEARIVLEANVMEILGIAINRYNGLTLRGVTMRPTSLAQRNE
MFFMCLDMMLSAAGINVGPISPDYTQHMATIGVLATPEIPFTTEAANEIARVTGETSTWG

vp7_btv1s(3)    TIAARALTVMRACATLQEARIVLEANVMEIL
vp7_btv10(5)    --AARALTVMRACATLQEARIVLEANVMEIL

vp7_btv1s       GIAINRYNGLTLRGVTMRPTSLAQRNEMFFM
vp7_btv10       GIAINRYNGLTLRGVTMRPTSLAQRNEMFFM

file.pdb

Contacts can be further analysed looking to the figure produced by ESPript:

file.2st

• DSSP¹⁵ (a PDB file can be directly uploaded if you use webESPript, DSSP being executed on the server)
• STRIDE¹⁶
• PHD¹⁷ (enter the full mail from Predict Protein)

 Warning: DSSP residue M does not match seq residue D 2 sequence 1 column 2

 Warning: DSSP residue M does not match seq residue D 2 sequence 1 column 2

 Warning: DSSP residue D does not match seq residue T 3 sequence 1 column 3

...........................

 Error: sec. structure elements are certainly misplaced