🧬 Protein Pathfinder User Guide

📖 Overview

Protein Pathfinder is a mobile application that helps researchers explore, validate, and visualize protein-protein interaction pathways. It searches for interaction pathways between two proteins using data from UniProt, STRING, PDB, and related evidence resources, and displays 3D molecular structures, phosphorylation sites, external evidence links, STRING network graphs, and validation information.

🚀 Quick Start

1. Search for Protein Interactions

1. Enter Gene Names
   • Type the first gene name (e.g., TP53)
   • Type the second gene name (e.g., EGFR)
   • Both fields support standard gene symbols
2. Choose a Network Type (optional)
   • Tap the network type dropdown (default: Functional)
   • Pick Physical, Regulatory (v12), or Regulatory (v12.5) if you need a different kind of evidence — see Network Types below
   • Your selection applies to the pathway search and to every evidence detail page you open from the results
3. Start Search
   • Tap the "Find Pathways" button
   • Wait 5-10 seconds while the app queries scientific databases
4. View Results
   • The app displays all discovered interaction pathways
   • Each pathway shows intermediate proteins (if any)
   • Evidence scores indicate interaction reliability

🌐 Network Types: Functional, Physical, Regulatory

STRING can return several different kinds of evidence for the same pair of proteins, depending on which network type you request. The app lets you choose one before searching, and you can change it again on any evidence detail page.

Network Type	What it means	Best for
Functional	Broadest evidence type. Includes indirect functional association — proteins that work in the same pathway or process, even without physical contact.	General exploration, default choice
Physical	Restricted to evidence for direct physical binding between the two proteins (complex formation, direct contact).	Structural questions, "do these two actually touch?"
Regulatory (v12)	Directional regulation (activation/inhibition) as served by the STRING v12.0 stable API.	Legacy comparison — see caveat below
Regulatory (v12.5)	Directional regulation with an explicit source → target arrow and activation/inhibition sign, served by STRING's v12.5 preview API.	Signaling direction — "does A activate or inhibit B?"

How to tell if you got real directional data

When STRING returns genuine directional evidence, the STRING Evidence page shows a colored arrow card above the score list:

• Green ▲ A → B (activation)
• Red ▼ A → B (inhibition)

If this card does not appear, the response had no direction/sign attached — this is expected for Functional and Physical, and is the known behavior described above for Regulatory (v12).

📊 Understanding the Results

Pathway Cards

Each pathway card displays:

• Route Description: The interaction path (e.g., "via MDM2-PIK3CA")
• Depth: Number of intermediate proteins
• Evidence: STRING database confidence score (0-100%)

Evidence Score Interpretation

Evidence Range	Meaning	Confidence Level
90-100%	Highest confidence	Strong experimental evidence
70-90%	High confidence	Multiple evidence sources
40-70%	Medium confidence	Some evidence exists
15-40%	Low confidence	Weak evidence
<15%	Very low	Minimal or no evidence

🔬 Viewing Protein Details

Opening Protein Information

1. Tap any protein card in the pathway
2. View comprehensive protein data:
   • UniProt ID: Official protein identifier
   • Gene Name: Standard gene symbol
   • Description: Protein function summary
   • Function: Detailed biological role
   • Location: Subcellular localization
   • Tissue Specificity: Where the protein is expressed

Phosphorylation Sites

• Orange cards show phosphorylation sites
• Each card displays:
  • Amino acid type (e.g., Ser, Thr, Tyr)
  • Position in sequence
  • Functional description

🧪 Cell Validation

Cell Validation helps you review whether a candidate pathway is biologically plausible in a selected cell context. When available, the app displays cell-related evidence for pathway proteins, including expression relevance, cell-type support, and validation notes from the integrated validation data.

How to Use Cell Validation

1. Open a pathway result.
2. Tap a protein or edge in the pathway.
3. Select Cell Validation from the detail/evidence menu.
4. Review the cell-specific evidence table and validation comments.

🧬 PhosphoSite Validation

PhosphoSite Validation checks whether phosphorylation sites such as TP53@S312, JUN@S63, BAD@S136, or MDM2@S288_T286 are supported by phosphorylation-site evidence. This is especially useful when a pathway is generated from site-aware search results.

What the Validation Shows

• Gene and residue: Protein name and phosphorylation position.
• Site support: Whether the phosphorylation site is found in the validation source.
• Evidence type: In vivo, in vitro, curated, predicted, or database-derived evidence when available.
• Kinase/substrate context: Known or inferred upstream kinase information when available.
• Confidence note: Practical interpretation of the site evidence.

🔗 External Evidences and Clickable Links

The app now provides an External Evidences section for proteins and edges. When a link is shown, tap it to open the corresponding evidence page or graph. Supported resources may include UniProt, STRING, STITCH, PDB/RCSB, and other pathway or validation resources depending on the available data.

Clickable Evidence Links

UniProtProtein metadata, sequence, function, subcellular location, and annotation. STRINGProtein interaction network and confidence evidence. STITCHChemical-protein interaction and inhibitor-related evidence. RCSB PDBExperimentally determined 3D structures and structure metadata.

STRING Direct Graph View

When viewing STRING evidence, the app uses a direct STRING network image when possible. This avoids blank screens caused by interactive STRING web pages that require browser-specific scripts, session handling, or plugin-style display behavior.

https://string-db.org/api/highres_image/network?identifiers=TP53&species=9606&add_white_nodes=15&network_flavor=confidence

The graph image can be viewed directly, zoomed, and compared with the current pathway. The external browser button can still be used to open the full STRING website when interactive browsing is needed.

🎨 3D Molecular Visualization

Viewing 3D Structures — Independent Dual-Panel Viewer

1. Tap the 3D viewer at the bottom of the screen
2. The viewer shows two separate panels, each with its own camera:
   • Left panel (purple): First protein
   • Right panel (blue): Second protein

Interacting with 3D Models

• Rotate: Drag with one finger — touch the left panel to rotate only the first protein, the right panel to rotate only the second
• Zoom: Pinch gesture, independently per panel
• Reset View: Tap the refresh button (top toolbar) — clears both panels and reloads both proteins from scratch, including turning off any electrostatic surface

Showing Phosphorylation Sites

1. Tap the P-sites button (scatter-dot icon) in the toolbar
2. Red spheres appear at phosphorylation sites, on whichever protein(s) have them
3. Button highlights when active
4. Tap again to hide sites

⚡ Electrostatic Surface (Approximate)

Tap the lightning bolt (⚡) button to color each protein's molecular surface by approximate electric charge: red for negative, blue for positive, white for neutral. This is computed instantly on your device using real CHARMM27 force-field partial charges for every atom of the standard 20 amino acids — not just the handful of charged residues used in earlier versions — combined with a Debye-Hückel distance screening model (V = Σ qᵢ/r · e^−r/λ) so nearby charges influence the surface more strongly than distant ones, similar to how ions are screened in solution.

Adjusting the Approximation

When the electrostatic surface is on, a small tune (🎚) icon appears next to the lightning bolt. Tap it to open a settings sheet with four sliders:

Parameter	What it controls
His charge	Blends histidine's side chain between its neutral and fully-protonated CHARMM27 charge sets (0 = fully neutral, 1 = fully protonated). Raise this to approximate a low-pH environment.
Contrast (clamp)	The potential value that maps to full red/blue saturation. Lower values increase visual contrast.
Debye length	How far a charge's influence spreads across the surface, in Ångströms. Lower = more localized "hot spots"; higher = broader, smoother regions.
Visual scale	An overall multiplier on the computed potential, for fine-tuning contrast.

The status bar briefly shows the actual computed potential range for each protein (e.g. ES: A[-3.2, 5.1] B[-2.8, 4.4] clamp±6.00) so you can see whether your settings are saturating the surface or leaving it too faint.

🧩 Understanding Pathways

Direct Interactions

Protein A ──────────> Protein B
Depth: 1
Evidence: 95%
        

The proteins interact directly with very strong evidence.

Indirect Interactions

Protein A ──> Intermediate ──> Protein B
Depth: 2
Evidence: 65%
        

The proteins interact through one intermediate protein. Evidence score reflects the reliability of both connections.

Complex Pathways

Protein A ──> X ──> Y ──> Protein B
Depth: 3
Evidence: 45%
        

Longer pathways have lower evidence scores due to multiple intermediate steps and cumulative uncertainty.

💡 Tips for Best Results

Choosing Proteins

Interpreting Evidence Scores

• High scores (>70%): Well-validated interactions, suitable for research planning
• Medium scores (40-70%): Plausible interactions, require experimental verification
• Low scores (<40%): Speculative interactions, use with caution

🔍 Example Searches

Cancer Research

• TP53 → EGFR
• TP53 → MDM2
• BRCA1 → BRCA2

Signal Transduction

• EGFR → AKT1
• AKT1 → MTOR
• MAPK1 → FOS

DNA Repair

• BRCA1 → RAD51
• TP53 → ATM

🛠️ Troubleshooting

"Could not identify proteins"

Cause: Gene name not found in UniProt database

Solution:

• Verify gene symbol spelling
• Try alternative gene names
• Ensure it's a human protein
• Check official HUGO gene nomenclature

"No pathways found"

Cause: No known interaction pathway in databases

Solution:

• Try closely related proteins
• Check if proteins are in same biological system

"3D viewer shows nothing"

Cause: No PDB structure available for the proteins

Solution:

• Not all proteins have crystallized 3D structures
• This is normal for many proteins
• Pathway information is still valid

"Changing the electrostatic settings sliders doesn't seem to do anything"

Cause: The default contrast (clamp) value may already be saturating most of the surface to full red/blue, or the view hasn't refreshed.

Solution:

• Check the status bar message after adjusting a slider — it shows the actual computed potential range (e.g. clamp±6.00) so you can confirm the update reached the viewer
• Try raising the Contrast (clamp) value if the surface looks entirely saturated, or lowering it if the surface looks entirely white
• Turn the electrostatic surface off and back on (⚡) to force a full recomputation

"Regulatory (v12) doesn't show a direction arrow"

Cause: This is expected behavior, not a bug — see the technical caveat in Network Types. STRING's stable v12.0 domain does not currently return directional data even when regulatory is requested.

Solution: Switch to Regulatory (v12.5) to see real direction and activation/inhibition sign.

"STRING graph opens but becomes blank after tapping"

Cause: Some STRING interactive web pages rely on browser scripts, session URLs, or plugin-style display behavior that may not render reliably inside the app.

Solution:

• Use the direct STRING graph image view inside the app.
• Use the external browser button for the full STRING website.
• Avoid relying on session-based cgi/network?taskId=... links for in-app display.

"Text is hard to read in night mode"

Cause: System dark mode may reduce contrast for some embedded web or evidence pages.

Solution: Use the app's high-contrast/light-mode display setting when available. The updated guide and evidence pages are designed with stronger black/white contrast.

📚 Data Sources

Database	Purpose	URL
UniProt	Protein metadata, sequences, annotations	uniprot.org
STRING	Protein interaction evidence scores (Functional / Physical / Regulatory v12)	string-db.org
STRING v12.5 (preview)	Directional regulatory evidence (source, target, activation/inhibition sign)	preview.string-db.org — pre-release, subject to change
PDB	3D molecular structures	rcsb.org
STITCH	Chemical-protein interaction and inhibitor-related evidence	stitch.embl.de
PhosphoSite / PTM resources	Phosphorylation-site validation and residue-level evidence	Available when supported by the app data source
Cell validation data	Cell-context support for pathway interpretation	Integrated validation table
3Dmol.js	3D visualization library	3dmol.org

🔐 Privacy & Data Usage

• No data stored: All searches are performed in real-time
• No personal information: App doesn't collect user data
• Free to use: No subscriptions or in-app purchases
• Academic use: Designed for research and educational purposes

📖 Glossary

• Gene Symbol: Standard abbreviation for a gene (e.g., TP53, EGFR)
• UniProt ID: Unique protein identifier (e.g., P04637)
• PDB ID: 3D structure identifier (e.g., 1A1U)
• Phosphorylation Site: Amino acid that can be chemically modified
• Evidence Score: Confidence level from STRING database (0-100%)
• Pathway Depth: Number of steps between proteins
• Intermediate Protein: Protein connecting two other proteins
• Cell Validation: Cell-context evidence used to judge whether a pathway is plausible in a selected biological setting
• PhosphoSite Validation: Residue-level check of phosphorylation sites such as S, T, or Y positions
• External Evidences: Clickable evidence links to UniProt, STRING, STITCH, PDB, or other supporting resources
• STRING Direct Graph: A direct image-based network view that avoids unreliable in-app rendering of interactive STRING pages
• Network Type: The kind of STRING evidence requested — Functional (broad association), Physical (direct binding), or Regulatory (directional activation/inhibition)
• Regulatory (v12) vs (v12.5): Two ways to request directional evidence; only v12.5 (STRING's preview API) currently returns real source/target/sign data — see Network Types
• Electrostatic Surface: A molecular surface colored by electric charge — red (negative), white (neutral), blue (positive)
• Partial Charge: The fraction of an elementary charge assigned to an individual atom by a force field, reflecting how electron density is distributed within a molecule
• CHARMM27: A widely-used molecular-mechanics force field; the source of the per-atom partial charges used to build the app's electrostatic surfaces
• United-Atom Charge: An atom's partial charge with any attached hydrogen's charge folded in — used here because most X-ray-derived PDB structures don't include hydrogen atoms
• Debye-Hückel Screening: A distance-based decay model (V = Σqᵢ/r·e⁻ʳ/λ) approximating how dissolved ions weaken a charge's influence over distance; the app uses it to spread each atom's charge across the surface