from the Stephen White laboratory at UC Irvine

MPEx Version 3.3 Full Documentation

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Plotting Region

The plotting region is the area with horizontal and vertical red lines. The number of residues is plotted on the x-axis, the hydropathy (or other analysis data) is plotted on the y-axis. The x-axis cannot be resized and is set to display 400 residues. When analysis plots of proteins with more than 400 residues are calculated, the axis will rescale automatically to display the entire sequence.

Hydropathy Analysis Plot legend (upper left corner)

"Smoothed Hydropathy" is obtained by sliding-window smoothing of hydropathy plot using a 5 AA window.

"Predicted TM Segments" are provided by MPEx in the "Locate" mode.

"Known TM Segments" appear for sequences loaded from MPtopo.

"Hydrophobic Moment" selectively displayed using check box on control panel.

"User Marked Segments" can be marked and measured using the "Mark/Measure User Segments" tool under MPExToolBox.

Translocon TM Analysis plot legend (upper left corner)

When the Translocon TM Analysis tab at the top of the plotting region is selected, MPEx reconfigures itself to do a screening analysis for the identification of transmembrane-favorable regions of an AA sequence based on translocon-mediated transmembrane helix assembly, considering amino acid position-dependent membrane insertion efficiency, as well as transmembrane segment length and flanking amino acid influences. The "full biological" hydrophobicity scales used for this analysis are from the supplemental information in Hessa et al. 2007 (Nature 450:1026-1030). A new set of legends will appear at the upper left of the plotting region, the y-axis label changes to "Predicted ΔG (kcal/mol)", and the control panel on the right-hand side changes.

Color lines followed by something like "L=23" represent the free energy analysis data for a sliding window of length 23.

"Predicted TM Segments" are favorable sequence regions whose lengths correspond to a window size within the range selected on the Translocon TM Analysis control panel.

"Known TM Segments" and "User Marked Segments" are as for the Hydropathy Analysis Plot legend.

If plot visibility for hydrophobic moment is selected in the control panel, a legend for this data plot will appear here, with an indication of the sliding window size it corresponds to.

β-Barrel Analysis Plot legend (upper left corner)

When the β-Barrel Analysis tab at the top of the plotting region is selected, MPEx reconfigures itself to do a screening analysis for the identification of β-barrel membrane proteins. A new set of legends will appear at the upper left of the plotting region, the y-axis label changes to "β-Strand/Hairpin Score", and the control panel on the right-hand side changes.

"β-Strand Scores" are obtained using a sliding-window representing the transmembrane window length (default 10 AA). A score is calculated for each window based on the two abundance data sets (interior and exposed) determined for β-barrel membrane proteins as described by Wimley in Protein Sci. 11:301-312( 2002).

"β-Hairpin Scores" are obtained by using a composite window of the hairpin window and its two adjacent transmembrane windows. A sum is calculated from the highest β-strand score of the two transmembrane windows. The β-hairpin score is thus highest when there are two β-strand peaks separated by a short loop.

"Predicted TM Regions" are those regions in the β-strand score plot with Y-values between 2 and 6, which is the range in which most membrane-spanning β-strands are found.

"Predicted Hairpin Regions" are those regions of the β-hairpin scores with values greater than 6. This cutoff value was found to identify correctly ~90% of the β-hairpins in the structure database (described in the article) without including many false peaks.

"Known TM Segments" and "User Marked Segments" are as for the Hydropathy Analysis Plot legend.

Residue Legend (lower left corner)

Classes of residues can be marked on the plot using the "Mark Residues" tool in the MPExToolBox dropdown. In Hydropathy Analysis mode, the Neutralized (d,e,h) legend item appears automatically whenever an H, D, or E residue is neutralized.

Cursor (Blue Cross-Hair)

The cursor is used to select or identify specific residues. It can be moved with the Mouse, by using the left/right arrow keys, or by using the "Send Cursor To" button on the Control Panel. To change the window focus to the plot region: with the mouse arrow in the plot window, click a mouse button.

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Data Buffer Control Bar

The Data Buffer Control Bar allows any analysis mode with valid data, including all selected parameter options, to be stored in a temporary storage buffer for later recall: Press the "Save to Data Buffer" button. The "List Buffers" button produces a list of the contents of buffers. The three menu areas on the right side of the bar (Load #, Delete #, and Comment #) allow quick buffer management. These regions change shade slightly when the mouse arrow is on them. The "Comment #" allows descriptive comments to be associated with a selected buffer.

Analysis modes with valid data that can be saved to a buffer are marked with a small green check mark () in the analysis mode selection tabs. Changing a feature of the sequence you are examining, esp. changing a residue, may invalidate analysis data for analysis modes other then the current one. Revisiting an analysis mode will revalidate the data for that mode.

Temporary storage buffers can also be manipulated using the "Buffers" menu under MPExToolBox (see below).

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Control Panel (Hydropathy Analysis)

Summary Window:

Scale

Current Wimley-White scale being used for calculations.

Residue at Cursor

Displays the residue at the cursor position. The cursor can be set by clicking the mouse within the plotting region. Dragging the mouse within the plotting region will move the cursor and change the display to indicate the current cursor position.

Partitioning

The current direction of partitioning used for calculations (bilayer-to-water or water-to-bilayer).

ΔG for Window

The total free energy calculated for the amino acid segment in the current window.

Hyd. Moment

The hydrophobic moment for the amino acid segment in the current window.

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Set Parameters:

Send Cursor To

Allows the user to position the cursor at a particular residue of interest. Type in the number of the residue at which you want the cursor placed. Press the button.

Select Wimley-White Scale

Three predefined hydropathy scales are available:

• Interfacial (IF): Using an experimentally determined hydrophobicity scale for amino acids at membrane interfaces, this option displays segments that are favored to partition into the interfacial region of the bilayer. For more information, see "Experimentally determined hydrophobicity scale for proteins at membrane interfaces," Nat. Struct. Biol. (1996) 3: 842-848 and Annu. Rev. Biophys. Biomol. Struct. (1999). 28: 319-365.
• Octanol (Oct): Determined by bulk-phase partitioning (i.e., partitioning to n-octanol), this option displays segments favored to associate in the hydrocarbon (non-polar) region of the bilayer. For more information, see Annu. Rev. Biophys. Biomol. Struct. (1999) 28: 319-365. This option is the default. We have recently shown that this scale allows one to identify TM segments with high accuracy. See Jaysinghe et al. (2001), J. Mol. Biol. 312: 927-934.
• Oct-IF (Octanol minus Interfacial): Calculates the free energy of partitioning to the hydrocarbon core minus the free energy of partitioning to the interface. Those segments on the interface that are thermodynamically favored to become transmembrane helices relative to the unfolded state in the interface are then displayed.

You may also select Other, which allows the use of a user-defined scale. The file chooser that appears with this selection shows an example of the required syntax for a scale-definition file.

Plot Visibility: Hydrophobic Moment

A toggle selection allowing a plot of the hydrophobic moment of the current sequence to become visible.

When the hydrophobic moment data are visible, calibration marks appear on the right-hand plotting region boundary. These calibration marks represent hydropathy values for an amino acid sequence like LLRRLLRRLRRLLRRLLR, where the two represented amino acids reside on opposite sides of an alpha-helix. (You can visualize this by copying this sequence into the Totalizer tool and viewing the helical wheel). Calibration marks are calculated for leucine and arginine (as above), leucine and lysine, leucine and serine, leucine and glycine, and leucine and aspartate. Depending on the visible range of the plot, not all of these marks may be visible.

The hydrophobic moment data can also be saved by using the "MPExFile->Save Data->as tab delimited text file" selection.

Select Mode

Hydropathy analysis may be run in one of two modes:

• Locate (default mode): After determining the hydropathy plot for the entered sequence, MPEx determines likely transmembrane segments that are a minimum of 19 residues in length. In this mode a window length of 19 residues is always used. The algorithm used is described in Jaysinghe et al. (2001), J. Mol. Biol. 312: 927-934.
• Scan: After determining the hydropathy plot for the entered sequence, MPEx determines those regions of the sequence favorable for partitioning into the membrane. In this mode, the user may select the length of the window used in the hydropathy plot.

Set Window Length

Sets the length of the sliding window used to calculate the hydropathy plot (scan mode only).

For example, with a Window Length of "19", the hydrophobicity of the first 19 residues (1-19) will be summed, and this sum is then assigned to the middle (10th) residue. Then, the hydrophobicity of the next 19 residues (2-20) will be summed and assigned to the middle (11th) residue, and so on. The window may contain an odd or even number of residues, but an odd number preserves symmetry around the central amino acid. Due to the fact that bilayers are typically 30 Å thick, to determine α-helical TM segments (at 1.5 Å rise/residue), approximately 20 amino acids are needed -- thus the default is 19. For TM β-strands (at 3.3 Å rise/residue) approximately 9 AA are needed.

Residues in Window

Displays the residues contained within the sliding window centered at the current cursor position. The residue at the cursor position is highlighted in red.

Send to Totalizer

Allows the user to analyze the energetics of the bilayer interactions of the residues in the sliding window using the Totalizer modules (see below). The current Wimley-White scale being used for analysis is inherited by the Totalizer.

Set Salt Bridges

Allows the user to form a salt bridge between two residues. This feature is useful for investigating the effect on the hydropathy plot of forming a salt bridge between a positively charged residue and a negatively charged one. When this option is selected, the user is presented with the Salt Bridges window. The user may then enter the sequence positions of the residues involved in a salt bridge. The hydropathy plot will be updated to reflect the changes in hydrophobicity resulting from the formation of the salt bridge. The salt bridge hydrophobicity scale and its rationale have been described in Jaysinghe et al. (2001), J. Mol. Biol. 312: 927-934.

Change Charge

Allows the user to change the charge of a single Asp, Glu, or His residue. Selecting this option will change the charged state of the residue at the cursor if there is an Asp, Glu, or His at this position. The hydropathy plot is automatically recalculated. The residue displayed in the Residue at Cursor box will change to reflect the charged state. Neutral Asp, Glu, or His residues are denoted by their lowercase single letter code (d, e, h). Charged residues are denoted by their UPPERCASE single letter code (D, E, H).

Change Residue To

Allows the user to change the residue at the cursor to any desired amino acid, and then recalculate the plot. Type in its ONE LETTER CODE. Changed residues are listed in the Results Window.

Restore Changed Residues

Restores all changed residues to their original values. This does not apply to residues whose charges have been changed by the Change Charge tool.

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Control Panel (Translocon TM Analysis)

Summary Window:

The summary window entries for Translocon TM Analysis are the same as those for the Hydropathy Analysis summary window, except that there is no Scale entry, and the entries for ΔG for Window and Hyd. Moment show what sliding window size their values correspond to (selected with Cursor on Data Set for Window control).

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Set Paramaters:

Send Cursor To

As for Hydropathy Analysis (see above).

Cursor On Data Set for Window

Translocon TM Analysis allows displaying data sets for an arbitrary number of sliding window sizes. Data values displayed in the "Summary Window" and "Residues in Window" display areas, as well as the hydrophobic moment plot, if visible, correspond to the data set the cursor is currently attached to. This can be adjusted to correspond to a size within the "Window Size Range" currently selected.

Plot Visibility: Hydrophobic Moment

Similar to Hydropathy Analysis, a toggle selection allowing a plot of the hydrophobic moment of the current sequence to become visible. The values displayed correspond to the data set the cursor is currently attached to. The moment is calculated using the "biological scales" used in Translocon TM Analysis.

When the hydrophobic moment data are visible, calibration marks appear on the right-hand plotting region boundary

The hydrophobic moment data for any, or all, of the window sizes in the selected range can also be saved by using the "MPExFile->Save Data->as tab delimited text file" selection.

Window Size Range

Sets the range of sliding window sizes used to calculate free energies.

Similar to window size selection in Hydropathy Analysis (see above), free energies for the residues in a window of a particular length are calculated and the value assigned to the middle residue within the window. The "biological scale" assigns individual residue free energy values based in part on a residue's relative position normal to a lipid bilayer.

A maximum and minimum size may be selected. Data plots for all window sizes within this range are generated. Predicted transmembrane segments will fall within this range.

Residues in Window

Displays the residues contained within the sliding window the cursor is currently attached to, centered at the current cursor position. The residue at the cursor position is highlighted.

Send to Totalizer

Allows the user to analyze the energetics of the bilayer interactions of the residues in the sliding window using the Totalizer modules (see below). The current window size that the cursor is following and the Translocon-TM hydropathy scale are inherited by the Totalizer.

Change Residue To

As for Hydropathy Analysis (see above).

Restore Changed Residues

As for Hydropathy Analysis (see above).

Visit DGpred at Stockholm U.

Takes your Web browser to a tool that does similar server-based analysis at Stockholm University.

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Control Panel (β-Barrel Analysis)

Summary Window:

β-Barrel Sequence Score

The β-barrel sequence score is the sum of all β-hairpin scores greater than 6 divided by the number of amino acids in the sequence. Based on an experimental set of known β-barrel membrane proteins, sequence scores of 2.0 and greater fall within the 97th percentile, whereas the median genomic score is 0.4. Sequence score values greater than the threshold value of 2.0 will appear in red in the summary window.

β-Strand Peaks

Known β-barrel structures contain an even number of between 8 and 22 β-strands and include monomeric, dimeric, and trimeric β-barrels. MPEx identifies the number of regions in the β-strand scores that exceed a threshold value of 4.0.

TM and Hairpin Window Scores

β-barrel analysis relies on a set of three adjacent sliding windows representing a potential transmembrane region, followed by a potential hairpin region, followed by a second potential transmembrane region. The cursor position represents the center of the hairpin window.

The transmembrane window score is calculated from a table of values representing the observed abundance of the amino acids and the architecture of known β-barrels of membrane proteins. Two values are calculated for each window: A score is the sum of the abundance values for interior (in) and exterior (out) alternating residues in the window. A second score is calculated starting with exterior and alternating with interior values. The greater of these two values is assigned as the window score.

Separating the two transmembrane windows is the hairpin window. This window is a recognizer of β-barrel architecture, and is based on two surrounding transmembrane window scores. The score for the hairpin window is the sum of the larger transmembrane β-strand scores from each of the surrounding transmembrane windows. It is thus highest when there are two β-strand peaks separated by a short loop represented by this window.

Residue at Cursor

As for Hydropathy Analysis (see above). The cursor position represents the center of the hairpin window.

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Set Paramaters:

Send Cursor To

As for Hydropathy Analysis (see above).

Plot Visibility

Two options are available, and both may be selected at once. Selecting β-Strand Scores displays a plot of the β-strand scores, predicted transmembrane regions in the sequence, and known transmembrane segments if the sequence was loaded from MPtopo.

Selecting β-Hairpin Scores displays β-hairpin scores, predicted hairpin regions, and known transmembrane segments if the sequence was loaded from MPtopo.

Set TM Window Length

The size of the transmembrane windows may be set to arbitrary values. Among the known β-barrel proteins used to develop this analysis, the β-barrel rise per residue was found to be 2.7 ± 0.25 Å, or about 10 ± 1 residues across the membrane. The default transmembrane window length is therefore 10.

Set Hairpin Window Length

The size of the hairpin window may be set to arbitrary values. Based on the β-barrel proteins used to develop this analysis, about half of the segments connecting membrane-spanning β-strands were shorter than six residues. The default hairpin window length is 5.

Residues in Windows

Displays the residues in the adjacent sliding first transmembrane window, middle hairpin window, and second transmembrane window, centered on the current cursor position. The residue at the cursor position is highlighted.

Change Residue To

As for Hydropathy Analysis (see above).

Restore Changed Residues

As for Hydropathy Analysis (see above).

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Results Window

The results of calculations are displayed automatically in the Results Window every time an analysis plot is recalculated.

Using the buttons at the bottom of the Results Window, the user may either save the results to a text file using the Save button, or print the results using the Print button.

NOTE: These actions require that MPEx 'step out of the sandbox' or secure environment to which it is ordinarily limited (i.e., perform actions considered potentially unsafe) to interact with the local file system. In order to facilitate these actions and to instill user confidence in taking such actions, we have signed the MPEx code with a digital certificate. Some runtime environments may prompt the user to grant/deny permissions for the sensitive operations.

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Data Buffers and Overlays

Besides the data buffer controls under the MPExToolBox menu, there are several data buffer controls on the main window above the graphing region (the Data Buffer Control Bar):

List Data Buffers:

Same as MPExToolBox/Buffers/List Data Buffers.

Save to Data Buffer:

Same as MPExToolBox/Buffers/Save Work to Data Buffer.

Load:

The "Load #" label above the graphing region enables a pop-up menu that lists saved data buffers by number. Selecting a buffer from this menu allows the user to load a saved buffer and its saved state to the workplace. Any subsequent changes made to the workplace will not affect the state of the buffer from which the workplace analysis plot was loaded.

Delete:

The "Delete #" label above the graphing region enables a pop-up menu listing saved data buffers, by number. Selecting a buffer from this menu enables the user to delete permanently a buffer from the collection of saved buffers. The user may delete all buffers at once by using the "Delete All Data Buffers" selection in the MPExToolBox/Buffers menu.

Comment:

The "Comment #" label above the graphing region enables a pop-up menu that lists saved data buffers by number. Selecting a buffer from this menu enables the user to view, add, or modify a comment associated with the buffer.

Selecting the Data Buffer Overlays tab at the top of the plotting region allows multiple saved buffers to be viewed together and manipulated in order to compare and contrast them with one another.

The Control Panel on the right side of the graphing region provides a vertical menu bar of all saved data buffers. The menu regions change shade slightly when the mouse arrow is on them. Color swatches on each menu correspond to the display color in the plotting region for that buffer. Selecting a menu in this region enables a pop-up menu listing operations available for the selected buffer, as follows:

Bring To Front

Brings the plot of the selected buffer to the foreground of the graphing region.

Change Color:

Customizes the color of the plot for this buffer.

Hide/Show

Toggles the visibility of the plot for this buffer.

TTM analysis window size:

If a Translocon TM Analysis data set is selected, this menu item is enabled. An analysis data set for a particular window size may be selected for display.

Comment:

View, add, or change the comment for this buffer.

Delete:

Deletes this buffer permanently.

The buffer data selection tabs at the bottom of the plotting region allow the user to select which data set to use for comparisons of the saved buffers. If a Translocon TM Analysis data set is selected, the menu on the Control Panel will display the window size (e.g., "L=19") for the dislayed data set. If a particular analysis had not been done on a sequence when its buffer was saved, the menu on the Control Panel will display a "No Data" icon for that buffer when the buffer data selection tab corresponding to that analysis is selected.

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MPExTotalizer

The Totalizer module can be used to determine the transfer free energies of short peptides or MP segments, and to obtain helical wheel diagrams, assuming the peptide forms an α-helix.

Summary Window:

The Summary Window for the Totalizer provides numerical results for the selected amino acid sequence and parameters, including the sequence length, the partitioning direction, the free energy for the entire sequence, the free energy for a selected subsequence of length 18 (if "Draw 18" is selected, below the helical wheel display area), the hydrophobic moment for the entire sequence, and the hydrophobic moment for a selected subsequence.

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Set Parameters:

Select Scale

Totalizer uses the Wimley-White scales, the "biological scales" used in Translocon TM Analysis, or a user-defined hydropathy scale (selectable with the Other button). The default scale is inherited from the current setting in Hydropathy Analysis or Translocon TM Analysis if a sequence is sent from there, or the octanol scale otherwise.

If the Translocon TM scale is selected, CONH and end group selections are disabled and calculations are based on the window size selected below the helical wheel display area.

When Other is selected, a file chooser showing an example of the required syntax for a scale definition file appears.

Δ CONH Control

See Set ΔCONH Value

End Groups

The N- and C-terminal endgroups can be specified. The option No End Groups is convenient for computing the hydrophobicity of subsequences, such as those within MP sequences. The free energies of endgroups are based upon experimental determinations (Hristova and White (2005), Biochemistry 44:12614-12619). The approximate effect of helical secondary structure on partitioning can be estimated by choosing As helix and setting % helicity to the value desired. This feature works only when the interfacial scale is selected.

Partitioning

The current direction of partitioning used for calculations (bilayer-to-water or water-to-bilayer).

Helical Wheel

The helical wheel display area has two modes for hydropathy scales. If the Draw 18 box is checked, 18 residues at a time are shown in the wheel. Move along the sequence one residue at a time by pressing Shift Right (i.e., toward C-terminus) or Shift Left (i.e., toward N-terminus). If the box is not checked, then the helical wheel for the entire sequence is shown. The vector arrow, measured to the tip of the arrow head, is proportional to the hydrophobic moment. No arrow vector may appear if the effective moment is effectively zero (less than ~0.01).

When the Translocon TM scale is selected, the Draw 18 selection box and end group selectors are disabled, and the Window Size spinner is enabled. This window size is used for free energy calculations and dictates how many residues are displayed in the helical wheel.

Controls on the right-hand side of the helical wheel display allow modification of the presentation. The wheel can be projected from either the N-terminus or C-terminus perspective (the end termini on the wheel graphic are marked with an N or C to make this clear), rotated about its central axis, or its radius adjusted.

Enter Sequence

An area to enter or edit a sequence for analysis is located between the helical wheel display area and the parameter panel.

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Batch Processing

The Batch Processing module automates the analysis of multiple FASTA format protein sequences with a single configuration. Hydropathy and/or Translocon TM analyses may be selected. Parameters that are not specific to a single sequence (such as salt bridges) may be selected. Input files, result types, and an output destination must be selected. (Batch Processing now accepts multiple FASTA sequences in each file). Once all required configuration is done, the Go button on the right-hand side is enabled. Progress may be monitored in the area at the upper right-hand side of the window.

Selecting an analysis type will expand an area to allow parameterization of that analysis type. The selections correspond to those available for the dedicated major analysis modes. See those sections of the documentation for further information.

Selection of analysis result types determines the output files generated. Result summary files resemble the results window seen with the major analysis modes. The tab-delimited data sets selection results in a file like that for "MPExFile -> Save Data -> as tab delimited text file". A new window will open for this selection, allowing choices of what data should be written.

Selecting Write Sequence Prediction Data Sets allows writing a file with predicted transmembrane sequences or predicted loop sequences, e.g.:
    proteinName -------YLGGAVALIVGVAFWLLY----------ALAPLAIIPV...    (TM segments)
or
    proteinName MDAVATA-------------------RSLDGSPhQS----------...    (loop segments)
with a tab character separating the protein name from the results data.

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Menu Bar

The menu bar contains pulldown menus labeled: MPExFile, MPExToolbox MPExWindow, and MPExHelp. (On Mac OS X, these will be located on the system menu bar at the top of the screen).

MPExFile (see note)

Choices under this menu, described below:

Read File
Enter Sequence
Read Local
Save Session
Restore Session
Save Data
Print
Quit MPEx

Read File

Contains two options, allowing the user to read sequences directly from the MPtopo membrane protein database or the Swiss-Prot protein database. Selecting any option will open an additional window: the MPtopo or the Swiss-Prot file reader, either of which then allow the user to select a protein sequence for analysis.

Read file from MPtopo database:

Read file from the Swiss-Prot database:

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Enter Sequence

Allows the user to manually enter a sequence of interest. Selecting this option brings up the Enter Sequence window into which the user can enter, in UPPERCASE SINGLE LETTER code, the sequence to be analyzed.

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Read Local*

Contains options that allow the user to read files containing sequences from the local disk. Four file formats can be read from the local disk:

• A text file with only the sequence in UPPERCASE SINGLE LETTER CODE.
• A text file in the MPtopo database file format.
• A text file in the FASTA file format.

NOTE: If MPEx contains data in its workspace that has not been saved to a memory buffer, the user will be prompted for a course of action. The user may select to overwrite the data in the workplace with new data, select to save the data in the workplace prior to reading in the new data, or cancel the operation entirely.

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Save Session*

Allows the current work session to be saved, including all of its current state information, as a local file. Save with file name extension '.mpx'.

Restore Session*

Allows restoring a previously saved session from a file. Restoring sessions saved from different release versions of MPEx will probably not work. (Sessions must be restored to the same version of MPEx from which they were saved. If you have a session saved from a previous version that you wish to restore, please visit the MPEx archive).

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Save Data*

Contains options that allow results to be saved to the local disk. Two types of files can be saved:

• A tab-delimited text file containing the raw data for the data sets. A dialog window will appear, allowing the user to select data sets to save from analyses performed. The output file can be read into Origin™ or Excel™ in order to recreate the plots calculated by MPEx.
• A text file containing the results displayed in the Results Window.

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Print*

Contains options that allow the user to print the plots generated by MPEx. The following three options are available:

• Print the plotting region.
• Print the entire MPEx window.
• Print the results displayed in the Results Window.

Print jobs will go to a local printer supported by the local operating environment. If the plotting region is larger than the selected paper size and orientation, a warning message should appear. At this point the user may either cancel the print job or continue with the current settings.

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Quit MPEx

Quits the MPEx Application. On Mac OS X, this item will appear in the MPEx application menu.

*Note:

Most actions performed from these menus require that MPEx 'step out of the sandbox', or secure environment, to which it is ordinarily constrained (i.e., perform actions considered potentially unsafe) to interact with the local file system. In order to facilitate these actions and to instill user confidence in taking such actions, we have signed the MPEx code with a digital certificate. Some runtime environments may prompt the user to grant/deny permissions for the sensitive operations.

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MPExToolBox

Choices under this menu, described below:

Search MPtopo
Set Partitioning From...
Set ΔCONH Value
Reset Residue Axis
Set Salt Bridges
Set Asp, Glu, His States
Mark Residues
Mark/Measure User Segments
Change Sequence Description
Comment, Add/Edit
Buffers

Search MPtopo

Allows the user to query the MPtopo database that contains experimentally determined membrane proteins. The queries are conducted on a MySQL database maintained by the White lab. The results returned from the database are displayed in the Querier Results window. The user may then analyze a returned result sequence by typing in the number of the result and pressing the Send to MPEx button in the Querier Results window.

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Set Partitioning From...

In Hydropathy Analysis or Translocon TM Analysis, allows the user to set the partitioning from either the Water Phase to the Bilayer Phase or from the Bilayer Phase to the Water Phase. This results in 'flipping' the y-axis. The default is set to represent the calculated TM regions on the positive y-axis, which represents negative (favorable) free energies.

Set ΔCONH Value

In Hydropathy Analysis, allows the user to modify the cost of partitioning an H-bonded peptide bond in a helix. Moving the slider to the right increases the cost of partitioning the peptide bond into the membrane, thus making the transfer more unfavorable, while moving the slider to the left reduces the cost of partitioning, making partitioning more favorable. The slider increases or decreases the cost of peptide bond partitioning in increments of 0.05 kcal/mol. The additional cost of partitioning selected by the user is displayed within the box. By default, the CONH value is set to 0.0 kcal/mol. Our investigations of the MPtopo database have indicated that this value yields the highest prediction accuracy. Using this value, the MPEx prediction accuracy for the TM segments in the MPtopo 3D_helix set of MPtopo was found to be >99%. See Jaysinghe et al. (2001), J. Mol. Biol. 312: 927-934.

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Reset Residue Axis

Allows the user to reset the numbering of the Residue Axis (x-axis). This feature is useful when the user is investigating an out-of-sequence segment from a larger protein sequence. By default, MPEx will number the first residue--residue 1. If, however, the segment under investigation is within the range of residues 200-400, say, of a larger sequence, then the user may wish to reset the first residue to 200. When the Reset Residue Axis option is selected, a pop-up window named Reset X Axis will appear. Type in the new start value and press the OK button. The residue axis (x-axis) will be reset to start at the new user-entered value. All output will now be relative to this new start value.

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Set Salt Bridges

In Hydropathy Analysis, allows the user to form a salt bridge between two residues. This feature is useful when the user is interested in investigating the effect on the hydropathy plot of forming a salt bridge between a positively charged residue and a negatively charged one. When this option is selected, the user is presented with the Salt Bridges window. The user may then enter the sequence positions of the residues involved in a salt bridge. The hydropathy plot will be updated to reflect the changes in hydrophobicity resulting from the formation of the salt bridge.

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Set Asp, Glu, His States

In Hydropathy Analysis, sets the charged state of the aspartate (Asp), glutamate (Glu), and histidine (His) residues. Three amino acids are titratable: aspartic acid (D), glutamic acid (E), and histidine (H). At physiological pH, aspartic acid and glutamic acid are charged, while histidine is neutral. Thus, these are the default settings for these three residues. Checking a box corresponding to each of these amino acids tells the program to make that residue neutral. See also the Change Charge option in the Parameter Selection Control Panel.

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Mark Residues

Allows the user to highlight residues of interest on the hydropathy plot. Selecting this option brings up a new window containing a list of the 20 amino acids. Selecting one or more of the residues will mark all occurrences of the residue(s) on the plot.

In Translocon TM Analysis, the residues will be marked on the data set corresponding to the window size the cursor is currently associated with.

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Mark/Measure User Segments

Contains two options that allow the user to mark and measure the transfer energy of regions of interest in the current plot. The options are:

Mark: When the Mark/Measure option is selected, a pop-up window named Mark/Measure Helix Segments will appear. You may then enter directly into the appropriate boxes the start and end positions of the region you would like to mark. Press the Mark button. The program will then mark the indicated region within the plot. You may also use a mouse button to mark the region directly on the plot. The start and end positions will be indicated in the appropriate box within the Mark TM Segs window. After marking the TM segments, press the Done button.

The free energy values displayed in the Mark/Measure Segments window and the user marked segments information in the Results window correspond to the currently selected Wimley-White scale from Hydropathy Analysis or 'Other'.

NOTE: When a region is marked, the region will be listed in the results window.

Remove: With the mouse, selectively remove a marked segment by selecting an entry from the list at the bottom of the Mark/Measure Segments window, then press the Remove button.

Remove all: Clears all user-marked regions indicated within the plot.

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Change Sequence Description

When this option is selected, a pop-up window named Change Sequence Description will appear. Enter a description for the sequence under investigation. After entering the new name, press the Set button. The new description will appear in the Sequence Description field at the top of the plotting region. It will also appear in the Results Window.

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Comment Add/Edit

Allows the user to add a comment to the protein under investigation. When selected, the user is presented with the Comment Add/Edit window. The user may then enter a descriptive comment. When the Set button is selected, this comment will be associated with the protein under investigation. The comment will also appear within the Results window.

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Data Buffers

Allows the user to save analysis plots to memory storage buffers.

Save Work to Data Buffer: Allows the user to save the current state of the analysis plot to a storage buffer. When this option is selected, the current state of the analysis plot (workplace) is copied and saved to a buffer. Subsequent changes to the workplace will not affect the state of the saved buffer. The user may select as many buffers as available memory can accomodate. The number of buffers saved is listed above the graphing region. The user may also save the workplace to a buffer using the "Save to Data Buffer" button located above the graphing region.

Delete All Data Buffers: Allows the user to delete all saved buffers at once. Individual buffers may also be deleted from the "Delete #" label pop-up menu, located above the main graphing region, or from the "Data Buffer Overlays" panel.

List Data Buffers: Presents the "Data Buffer List" window to the user, listing descriptive information, including parameter settings for the analyses used, about saved buffers. The user may also list saved buffers with the "List Data Buffers" button above the main graphing region.

NOTE: The 'Save Work to Data Buffer' and 'Overlay Buffers' options can be used to compare multiple analysis plots of either the same sequence under different conditions, or the analysis plots of different proteins. For example, these options can be used to compare the hydropathy plots of homologous proteins (see above).

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MPExWindow

Choices under this menu, described below:

Results

View the results window.

Sequence

View the sequence under investigation.

Log

View a log of user operations performed.

Wimley-White Scales

View the Wimley-White hydrophobicity scales used in the hydropathy analysis calculations.

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MPExHelp

Contains options to display the Help documentation (this document) for MPEx, including version and copyright information. Links to reference publications for the different analyses are also available.

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Frequently Asked Questions (FAQ)

Can I save my current work and restore it later?
What happens if I try to restore a session saved with a previous MPEx version?
How do I enter my own sequence for analysis?
How do I interpret the data in the Results Window?
How do the three Wimley-White hydropathy scales differ from one another?
How can I find out if I have particular residues in my sequence and, if so, where they are located?
How can I find the total transfer of free energy for a part or all of my sequence?
How can I print the results for the protein I am analyzing?
I know which segments of my sequence are transmembrane. How can I directly compare them with those determined by MPEx?
How can I change the charge for a specific aspartic acid, glutamic acid, and/or histidine?
I know that some residues in my protein form salt bridges. Can I look at how these salt bridges change the hydropathy plot?
How can I compare the analysis plots of more than one protein?
Can I compare the analysis plot for a single protein under different conditions?
I saved several analysis plots to buffers, but I don't remember the conditions under which each was saved. What do I do?
I saved an analysis plot to a buffer. Can I change the saved plot?
What is this 'workplace' I keep reading about?
I have a sequence and I would like to put it into a MPtopo file format. How can I do that?
I have a sequence and I would like to put it into a FASTA file format. How can I do that?
What security issues affect running MPEx?
What are the browser and OS requirements to run MPEx?
Is it possible to run MPEx without a browser?

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Can I save my current work and restore it later?

Yes. You can save your work session to a .mpx file and restore it later, enabling you to continue working with the same session.

• To save a work session, choose 'Save Session' from the MPExFile menu. This will bring up a file dialog asking you where to save it.
• To restore a session, choose 'Restore Session' from the MPExFile menu. Saved session files will have a .mpx suffix.

Restoring a saved session will destroy the current work space. If you have work underway that you wish to revisit later, you should save it before doing a restore. A caution to this effect will be given when doing a session restore.

Note: New versions of MPEx become available periodically. Sessions saved with an older release of MPEx probably won't be compatible with a newer release. If you have sessions saved with a previous MPEx version that you need to access, check the MPEx archive for earlier releases.

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What happens if I try to restore a session saved with a previous MPEx version?

Because MPEx internal data structures are likely to change between versions, you'll probably get an error message like "Unable to restore session from ...". Previous versions of MPEx are archived here. You should be able to restore your session with the appropriate version of MPEx at the archive site.

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How do I enter my own sequence for analysis?

To enter your own sequence by typing it in:

• Choose 'Enter Sequence' from the MPExFile menu. A new window named 'Enter User Sequence' will appear.
• Enter your sequence by typing in the one letter codes in capital letters. You can also paste in a sequence directly.
• Click 'Enter'.
• A hydropathy plot will be calculated and displayed within the Plotting Region.

NOTE: Please make sure that you enter your sequence using the SINGLE LETTER UPPERCASE codes for the amino acids. Any letter not recognized as a valid single letter code will be automatically discarded before the hydropathy plot is calculated.

To enter your own sequence from a file on the local disk, you must first create a file on the local disk containing the sequence. The format of the file must be one of the following:

• A text file containing only the sequence in SINGLE LETTER UPPERCASE codes (neutral Asp, Glu, or His residues are denoted by their lowercase single letter code (d, e, h), charged residues are denoted by their UPPERCASE single letter code (D, E, H)).
• A text file formatted in the MPtopo file format.
• A text file formatted in the FASTA file format.

Choose 'Read Local' from the MPExFile menu and select the option for the file type you would like to read in. Follow the directions in the System File read dialog box.

NOTE: Reading a file from your local disk requires that MPEx 'step out of the sandbox', or secure environment, to which it is ordinarily limited (i.e., perform actions considered potentially unsafe). In order to facilitate these actions, we have signed the MPEx code with a digital certificate. Some browsers may prompt the user to grant/deny permissions for the sensitive operations.

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How do I interpret the data in the Results Window?

The MPEx window displays the following information:

• MPEx general settings, including the analysis mode the results correspond to, the filename of the peptide/protein, user comments, any residues that have been changed from the original sequence, any user marked segments, with their Wimley-White scale and free energy value, and the amino acid working sequence.
• Analysis specific settings. These include:

  For Hydropathy Analysis:

  Whether the titratable AAs are charged or not (and if not, which residues have been neutralized), any residues in salt bridges, Wimley-White scale used for calculation, ΔCONH setting, mode (Locate or Scan) in which the hydropathy plot was calculated, partitioning direction, and the window size.

  For Translocon TM Analysis:

  Partitioning direction and window size range.

  For β-Barrel Analysis:

  Transmembrane and hairpin window sizes.

• Common result features include: the number, position, length, and one letter codes of predicted transmembrane segments, known transmembrane segments, and user marked segments.
• Analysis specific results. These include:

  For Hydropathy Analysis:

  Free energy values for the predicted transmembrane segments (from the current scale) and free energy for the 19 amino acids centered at the peak value for the predicted segments (and the one letter codes of those). The free energies for any known segments and for user marked segments are also given.

  For Translocon TM Analysis:

  Free energy values based on the biological scale for predicted and any known transmembran segments. Free energy values (based on currently selected Hydropathy Analysis Wimley-White scale) for and use marked segments.

  For β-Barrel Analysis:

  A β-Barrel sequence score, the number of β-strand peaks, and the number of predicted hairpin regions. Free energy values for known transmembrane segments and user marked segments use the currently selected Wimley-White scale from Hydropathy Analysis.

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How do the three Wimley-White hydropathy scales differ from one another?

The hydropathy scales are based on experimentally determined whole-residue transfer free energies for each amino acid. The experiments involve partitioning to the POPC bilayer interface and into bulk-phase solvent (octanol, which imitates the hydrophobic core of a bilayer). The interface scale measures the free energy of transfer from water to the bilayer interface. The octanol scale measures the free energy of transfer from water to octanol. Therefore, if one were to subtract the octanol scale from the interface scale, one could see whether those segments that can partition on the interface would have favorable energy to become transmembrane helices. For additional information, see the Wimley-White Hydrophobicity Scale page.

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How can I find out about particular residues in my sequence and where they are located?

To view specific residues:

• Choose 'Mark Residues' from the 'MPExToolbox' menu. The Mark Residues window will appear.
• Select the residue you would like to mark from the list. You may choose more than one residue at a time.
• If the amino acid(s) is in the sequence, it will be marked on the plot.
• The residue markings are color-coded according to the legend at the bottom of the plot.

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How can I find the total transfer of free energy for a part or all of my sequence?

To calculate just the free energy of a sequence (Wimley-White scales):

• Choose 'Activate Totalizer' from the 'MPExToolbox' menu. The Totalizer module window will appear. This module acts as a calculator for transfer free energy.
• Enter (using the one letter AA codes) a part or all of your sequence. Choose the scale (Interfacial, Octanol, or Oct-IF) and press the 'Calculate' button. The results appear in the window beneath the entered sequence.
• Alternatively, the Mark/Measure User Segments tool, under the MPExToolBox menu can also be used.

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How can I print the results for the protein I am analyzing?

To print:

• Choose the 'Print' option from the 'MPExFile' menu.
• Select the desired printing option: graph only, full MPEx frame, or the Results Window.
• Follow the directions of the system print dialog.

You may also press the Print button from within the Results Window or Overlay Window to print their contents.

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I know which segments of my sequence are transmembrane. How can I directly compare them with those determined by MPEx?

MPEx allows users to mark their own TM segments:

• Choose 'Mark/Measure' in the 'Mark/Measure User Segments' submenu under the 'MPExToolbox' menu. The 'Mark/Measure Segments' window will be activated.
• Enter the Start and End positions of a desired segment. Press the 'Mark' button.
• Alternatively, once the 'Mark Helix Segs' window is activated, click and drag the mouse within the hydropathy plot. The Start and End positions will be displayed automatically. Press the 'Mark' button.
• A rust-colored bar appears, indicating the marked section.

The free energy values displayed in the Mark/Measure Segments window and the user marked segments information in the Results window correspond to the currently selected Wimley-White scale from Hydropathy Analysis.

To remove a single marked helix:

• Choose 'Mark/Measure' in the 'Mark/Measure User Segments' submenu under the 'MPExToolbox' menu. The 'Mark/Measure Segments' window will be activated.
• Select the marked segment you would like to remove from the list at the bottom of the window. Press the Clear button.
• The 'Clear all' option in the 'Mark/Measure Segments' window removes all the marked segments from the plot.

To remove all the marked segments: from within the 'MPExToolbox' menu, choose the 'Mark/Measure User Segments' submenu and select 'Clear All'.

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How can I change the charge for a specific aspartic acid, glutamic acid, and/or histidine?

To change the charge of a specific residue:

• Place the cursor on the amino acid whose charge is to be changed.
• Select the 'Change Charge' option from the Parameter Selection Panel.

NOTE: Charge can be changed only for Asp, Glu, and His.
Only Hydropathy Analysis is affected by any of these changes.

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I know that some residues in my protein form salt bridges. Can I look at how these salt bridges change the Hydropathy Analysis plot?

Yes.

• Press the 'Set Salt Bridge' button on the Parameter Selection Panel. OR select 'Set Salt Bridges' from the MPExToolbox menu.
• A new window named 'Salt Bridges' will appear.
• Enter the residue pair in the salt bridge into the textboxes.
• Press the 'Set' button.

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How can I compare the analysis plots of more than one protein?

To compare the plots of more than one protein:

• Select an analysis mode, then read in a protein. This calculates the data for the selected analysis.
• Save the plot to a buffer using the 'Save Work to Buffer' in the 'Buffers' submenu under the 'MPExToolbox' menu. OR press the 'Save' button above the plotting region.
• Read in another protein and save the analysis plot to a buffer.
• Select 'Overlay Buffers' in the 'Buffers' submenu under the 'MPExToolbox' menu. The 'Overlay Window' will appear showing the overlaid analysis plots of the two proteins.
• As you read in other proteins and save the analysis plots to a buffer, the overlay window will be updated to show the newly added proteins.

NOTE: This feature can also be used to compare analysis plots of a single protein under different conditions.

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Can I compare analysis plots for a single protein under different conditions?

Yes.

• After generating an analysis plot under a specific set of conditions, save the plot to a buffer using 'Save Work to Buffer', found in the 'Buffers' submenu under the 'MPexToolbox' menu. OR press the 'Save' button above the plotting region.
• Generate an analysis plot under a different set of conditions and save it to a buffer.
• Select 'Overlay Buffers' in the 'Buffers' submenu under the 'MPExToolbox' menu. The 'Overlay Window' will appear, showing overlaid analysis plots generated under the different conditions.

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I saved several analysis plots to buffers, but I can't remember the conditions under which each was saved. What do I do?

Press the 'List' button, found above the graphing region. The 'Buffer List' window will appear, with information about each buffer saved in memory. Consult this information to learn the conditions under which each buffer was generated.

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I saved an analysis plot to a buffer. Can I change the saved plot?

Yes.

• First, load the saved buffer you want to work on into the workplace. Enter the buffer number you want to work on in the textbox above the graphing region. Press the 'Load' button above the graphing region. The buffer will be loaded onto the workplace. (Note: If the workplace contains any data that is not saved you will be prompted for some action).
• Make changes to the plot.
• Press the 'Save' button above the graphing region to save the altered plot to a buffer. You will be presented with a dialog box. Follow the directions.

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What is this 'workplace' I keep reading about ?

The 'workplace' is the analysis plot and is displayed within the plotting region. Changes made from the Parameter Selection Panel or from the MPExToolbox will affect only the analysis plot in the workplace.

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I have a sequence, and I would like to put it into a MPtopo file format. How can I do that?

The MPTopo format is a text file containing Field Name:Field Value pairs. Using a text editor, create a text file with the following format:

protein_name: bacteriorhodopsin (h. salinarium)
file_name: bR.txt
entry_date: 27may98
refman_number: 17666
author: Luecke,H., Schobert,B., Richter,H.-T., Cartailler,J.-P., Lanyi,J.K. (1999) [Structure of bacteriorhodopsin] { J Mol Biol, 291, 899-911}
remarks: Sequence below is processed sequence from Swiss-Prot (13 AA signal sequence removed).
pir: RAHSB
Swiss_Prot_entry: BACR_HALN1
Swiss_Prot_number: P02945
Swiss_Prot_gene: BOP,VNG1467G
PDB_title: bacteriorhodopsin
PDB_Identifier: 1BRX,2BRD,1AP9,1AT9,1BRD
N_terminal: out
number_tmsegs: 7
tm_segments: A.9,31;B.36,63;C.81,101;D.104,127;E.133,155;F.164,192;G.200,225
sequence: QAQITGRPEWIWLALGTALMGLGTLYFLVKGMGVSDPDAKKFYAITTLVPAIAFTMYLSML LGYGLTMVPFGGEQNPIYWARYADK*

The field names in boldface gray -- protein_name, file_name, and sequence -- are required. The others need not be included for the file to be readable. Each field/value pair must be on one line (your browser may show wrapped lines that extend beyond the width of its viewing area). The sequence in this example has been shortened for illustration purposes.

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I have a sequence, and I would like to put it into a FASTA file format. How can I do that?

Create a text file in the following format:

>CCEG PIR1 release 62.08

GDAERGKKLFESRAAQCHSAQKGVNSTGPSLWGVYGRTSGSVPGYAYSN

NKNAAIVWEEETLHKFLENPKKYVPGTKMAFAGIKAKKDRQDIIAYMKTLKD

The first line may contain any characters you wish (or none at all following the required ">" first character), but it is usually the name of the protein followed by a description of the sequence. The remaining lines contain the sequence itself. Blank lines, whitespace characters, dashes, underscores and periods are ignored.

More detailed information on the FASTA format is available here.

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What security issues affect running MPEx?

MPEx code is signed using a digital certificate obtained from Thawte, Inc. When MPEx is run, the user will be prompted on security issues concerning the current operation and given an opportunity to examine the certificate information. At this time the user has an opportunity either to grant or deny permission for the operation. If the user grants the requested permission, MPEx will start normally. Accepting the security warning and allowing MPEx to run grants MPEx privileges that include:

• Connecting to the Swiss-Prot database: Requires MPEx to make an internet connection to an IP address other than that from which it originated and possibly download data from that site.
• Reading sequence files or restoring sessions from the local disk: Requires MPEx to read files from the local disk.
• Writing results or saving session to the local disk: Requires MPEx to write files to the local disk.
• Printing graphs and results: Requires access to the system printing resources.
• Saving or restoring sessions: Requires MPEx to read or write files to the local disk.

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What are the browser and OS requirements to run MPEx?

MPEx now runs with Java Web Start, which is part of the Java Runtime Environment. If you don't have Java installed on your computer, your browser should take you to a web site where you can download it when you click on the 'Start MPEx' button. Otherwise, if you don't have Java installed on your computer, or you don't have an up-to-date version (you should be running at least version 6 of the JRE), visit the Java download site, check the Help menu at the top of your browser for plugin information, or check with your system administrator.

We expect few problems if your Java environment is up-to-date, but please let us know if you experience strange behavior with MPEx.

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Is it possible to run MPEx without a browser ?

MPEx no longer runs within a browser as a Java applet but, rather, is downloaded as a Java program and run through Java Web Start, which is packaged as part of the Java Runtime Environment package (see just above if you need to acquire this).

Once you have downloaded MPEx, you can run it without using a browser, or even without being online, by using the Java Web Start console. Start the console with the command 'javaws -viewer' from a command or terminal window (just the command 'javaws' will show all the available options). From the console, you can run run any of the Java Web Start programs you have downloaded, create shortcuts to them, etc.

If you are online, Web Start should check for newer versions of any program you happen to run within Web Start.

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