Documentation › Overview
Overview
Predict protein compatibility with apoferritin nanocage encapsulation
enCAGE uses experimentally derived structural descriptors to estimate whether a protein is likely to undergo productive encapsulation inside apoferritin.
The challenge
Loading proteins into ferritin nanocages is still largely empirical. A cargo must be small enough to fit inside the ~8 nm cavity, yet compatible with the interior surface charge to avoid non-productive interactions. These factors are difficult to predict from sequence alone.
What enCAGE does
enCAGE analyses three physicochemical descriptors from a protein's 3D structure — steric compatibility, electrostatic complementarity and structural organisation — and applies a hierarchical decision framework calibrated on an experimental cargo panel.
What you receive
- Compatibility classification (Regime I, II or III)
- Descriptor values with interpretation
- Mechanistic explanation
- Downloadable report
How enCAGE works
Protein Structure
Upload a PDB file or enter a PDB ID.
Descriptor Extraction
Compute size, charge and structural organisation descriptors.
Decision Framework
Apply experimentally derived rules to determine the assembly regime.
Prediction Report
Receive prediction, descriptor values and mechanistic explanation.
Important
enCAGE is intended as a design-support tool rather than a validated classifier. Predictions should be interpreted alongside experimental validation and other considerations relevant to your system.
Experimental foundation
Calibrated on a panel of representative protein cargos with known encapsulation outcomes.
Transparent & interpretable
Rules and thresholds are explicit and biologically meaningful.
Reproducible
All calculations use open-source tools and documented parameters.
Privacy first
Structures are processed transiently — nothing is stored after analysis.
New to enCAGE? Start with Examples to see how predictions are made for known proteins.
Documentation › Understanding Results
Understanding Results
Learn what your prediction means and how enCAGE makes each call.
Overview: how enCAGE makes a prediction
enCAGE compares your protein with the properties of the ferritin nanocage and follows a simple sequence of checks.
1. Charge check
Is the surface strongly positively charged?
2. Size check
If charge-compatible, does it fit inside ~8 nm?
3. Flexibility check
If it's too large, could flexibility allow loading?
The answers determine which outcome is most likely.
The three assembly regimes
Depending on your protein's charge, size and structural features, one of three outcomes is predicted.
Your protein is compact and charge-compatible. During cage reassembly it is expected to be enclosed inside the nanocage.
Typical characteristics
- Fits within the ~8 nm cavity
- Charge is compatible (not strongly +)
- Usually compact or globular
What to expect
Reliable encapsulation with minimal disruption to cage assembly.
Your protein appears too large to fit based on its static structure, but flexible or multidomain proteins can sometimes reorganise during cage assembly.
Typical characteristics
- Charge is compatible
- Larger than the nominal cavity
- May be flexible or multidomain
What to expect
Encapsulation is possible, but should be confirmed experimentally.
A strongly positive surface charge causes the protein to bind to ferritin subunits instead of being enclosed inside the cavity.
Typical characteristics
- Strongly positive surface charge
- Electrostatics dominate
- May fit sterically
What to expect
Off-pathway assembly and poor encapsulation.
When no encapsulation is predicted
This outcome is returned when the protein is:
- Larger than the ferritin cavity (~8 nm)
- Not strongly positively charged
- Unlikely to rearrange during assembly (rigid, single-domain)
Unlike Regime II proteins, rigid single-domain proteins have no obvious way to fold or repack as the cage closes.
What to expect
Encapsulation is unlikely using standard ferritin reassembly. You may wish to explore:
- Smaller construct or truncated protein
- Split the protein into domains
- Use a different nanocage system
- Modify surface charge
Why isn't this called Regime IV?
Regimes I–III were each observed experimentally during calibration.
This fourth outcome follows from the logic of the framework when no feasible route to encapsulation exists for a rigid, oversized protein.
How the call is made
Is the protein strongly positively charged?
Does Dmax fit within ~8 nm?
Could flexibility allow accommodation?
Key thresholds
- Cavity diameter
- ~8 nm
- Cationic charge threshold
- +5 (pH 7.4)
- Flexibility
- Not measurable from static structure
Quick comparison
| Outcome | Charge | Size (Dmax) | Flexibility | Likelihood | Recommendation |
|---|---|---|---|---|---|
| Regime I Efficient luminal encapsulation |
Compatible (not strongly +) | Fits within ~8 nm | Any | High | Proceed with encapsulation |
| Regime II Accommodation possible |
Compatible (not strongly +) | Larger than ~8 nm | Likely flexible / multidomain | Moderate | Worth testing experimentally |
| Regime III Charge-driven off-pathway assembly |
Strongly positive | Any | Any | Low | Reduce net charge or use alternative strategy |
| No encapsulation predicted | Compatible (not strongly +) | Larger than ~8 nm | Rigid / single-domain | Very low | Consider smaller construct, split, or different cage |
Remember: these are predictions based on known rules and empirical thresholds. Experimental validation is always essential.
Documentation › Descriptors
Descriptors
How enCAGE interprets your protein
Each descriptor captures a different aspect of your protein's size, shape, or surface chemistry. Together, they help enCAGE predict whether a protein can be successfully encapsulated inside ferritin.
How enCAGE uses these descriptors
1 Net charge
Checked first (electrostatics dominate)2 Dmax
Steric fit within ~8 nm cavity3 Flexibility
Helps oversized proteins adapt4 Supporting descriptors
Refine the predictionNet charge and Dmax drive the call; the rest add context.
Descriptor categories
Size & Shape
How big and how shaped
Occupancy
How much space is used
Surface properties
Charge and interactions
Values are calculated from your protein structure at pH 7.4 unless stated otherwise.
Dmax
★★★★★ PrimaryWhat is it?
The single longest straight-line distance between any two atoms in your protein, in nanometres.
Why it matters
This is the strictest test of whether your protein can physically fit through the ~8 nm ferritin cavity.
Rule of thumb
- < 6 nm Fits the cavity (Regime I)
- ~ 6–8 nm Borderline (check flexibility)
- > 8 nm Too large unless flexible
Length × width × thickness
★★★★☆ SupportingWhat is it?
Your protein's bounding box along its three principal axes (longest to shortest), in nanometres.
Why it matters
Gives a sense of overall shape beyond Dmax alone. Helps identify elongated (rod-like) or flattened (disc-like) proteins.
Rule of thumb
No strict threshold. Use together with Dmax and shape anisotropy.
Volume ratio
★★★★☆ SupportingWhat is it?
The fraction of the cavity's internal volume your protein would occupy. The cavity is treated as a 268 nm³ sphere (~8 nm diameter).
Why it matters
Higher occupancy leaves less free space for water and cage flexibility, which can reduce loading efficiency.
Rule of thumb
- < 0.30 Low occupancy (favourable)
- 0.30–0.60 Moderate
- > 0.60 High occupancy (less efficient)
Example
Your protein occupies ~15% of the cavity.
Molecular volume
★★★★☆ SupportingWhat is it?
The physical volume of your protein itself, in nm³, before it's expressed as a fraction of the cavity.
Why it matters
Used to calculate volume ratio, and to compare cargos of different sizes on a common scale.
Rule of thumb
No fixed threshold. Compare relative to the 268 nm³ cavity.
Example
39 nm³
≈ 15% of cavity volume
Net charge (pH 7.4)
★★★★★ PrimaryWhat is it?
Your protein's overall surface charge at physiological pH, estimated using the Henderson–Hasselbalch approach.
Why it matters
Strongly positive proteins are attracted to the negatively charged cage exterior and may assemble off-pathway.
Rule of thumb
- < +3 Charge-compatible (Regime I or II)
- ~ +3–5 Near threshold (use caution)
- > +5 Likely off-pathway (Regime III)
Shape anisotropy (κ²)
★★★☆☆ SupportingWhat is it?
A number from 0 to 1 describing how far your protein departs from a perfect sphere.
Why it matters
Indicates whether a large Dmax reflects a genuinely big protein (high κ²) or just an elongated shape.
Rule of thumb
- < 0.3 More spherical
- 0.3–0.7 Moderately anisotropic
- > 0.7 Highly elongated (rod-like)
Remember: These descriptors are calculated from your protein structure and used together to make a prediction. They are not direct measurements and should be interpreted as guides, not absolute rules.
Documentation › Methods
Methods
How are the calculations performed?
enCAGE turns a single protein structure into six descriptors. These values follow a fixed hierarchy to assign one of the assembly regimes.
At a glance: how predictions are made
1 Net charge
Checked first (electrostatics dominate)2 Dmax
Steric fit within ~8 nm cavity3 Flexibility
Helps oversized proteins adapt4 Outcome
One of three regimes or no encapsulationThresholds are adjustable in the tool. See limits below.
The six descriptors
Each descriptor captures a different aspect of your protein. Select a card to jump to its details.
Dmax
Longest straight-line distance across the protein (nm).
Size & Shape 2Length × width × thickness
Overall dimensions along the protein's principal axes (nm).
Size & Shape 3Volume ratio
Fraction of the 268 nm³ ferritin cavity occupied by the protein.
Occupancy 4Molecular volume
Estimated physical volume of the protein (nm³).
Occupancy 5Net charge (pH 7.4)
Overall surface charge at physiological pH (Henderson–Hasselbalch).
Surface properties 6Shape anisotropy (κ²)
How elongated the protein is (0 = sphere, 1 = rod).
Size & ShapeHow each descriptor is calculated
Dmax
The largest pairwise distance between any two heavy atoms in the structure, computed directly.
Learn more
Very large structures (>6,000 heavy atoms) are randomly subsampled for speed, with a fixed seed so results stay reproducible.
Why it matters
This is the strictest test of whether your protein can physically fit inside the ~8 nm ferritin cavity.
Length × width × thickness
The protein is rotated onto its principal axes (via PCA on the coordinate covariance matrix). The extents along these axes define length (L), width (W) and thickness (T).
Why it matters
Gives a sense of overall shape beyond Dmax, and helps identify elongated (rod-like) or flattened (disc-like) proteins.
Volume ratio
Protein volume divided by the internal volume of the ferritin cavity (268 nm³, a sphere ~8 nm in diameter).
Why it matters
Higher occupancy leaves less free space for water and cage flexibility, which can reduce loading efficiency.
Example
Your protein occupies ~15%
Molecular volume
Estimated physical volume of the protein, in nm³, before any scaling.
Learn more
By default this is a fast grid-based van der Waals union approximation (0.9 Å spacing), which underestimates the true solvent-excluded volume since no probe is rolled over the surface. A ChimeraX solvent-excluded volume can be supplied under Advanced options instead.
Why it matters
Used to compute volume ratio, and to compare cargos of different sizes.
Example
39 nm³
≈ 15% of cavity volume
Net charge (pH 7.4)
Calculated using the Henderson–Hasselbalch equation, summing the fractional charge on ionisable residues at pH 7.4.
Learn more
Ionisable groups: N-/C-termini, Lys, Arg, His, Asp, Glu, Cys, Tyr, using a standard EMBOSS pKa set. Ignores local electrostatic environment and structural burial — supply a Prot pi value under Advanced options for a more rigorous estimate.
Why it matters
Strongly positive proteins tend to bind ferritin subunits and assemble via an off-pathway route (Regime III).
Cationic threshold: +5.0
Shape anisotropy (κ²)
Calculated from the same principal axes using the standard relative-shape-anisotropy formula (independent of overall size).
Why it matters
Helps judge whether a large Dmax reflects genuine bulk (high κ²) or an elongated shape that might still adapt.
The regime decision, and its limits
The descriptors feed into a fixed hierarchy: net charge is evaluated first, because a strongly cationic surface can override everything else and divert the cargo into off-pathway assembly (Regime III). Charge-compatible cargos are then sorted by Dmax against the ~8 nm cavity diameter into Regime I (fits) or, if oversized, resolved on structural organisation: multidomain or conformationally flexible cargos are assigned Regime II, while single-domain rigid cargos are reported as predicted non-encapsulating.
These thresholds — the default pH of 7.4 and the +5.0 cationic cutoff — are calibrated on a small in-house cargo panel (equine apoferritin, pH cycling) and are exposed as adjustable parameters in the tool rather than hard-coded.
Important things to know
-
Heuristic, not a classifier
enCAGE is a design aid. Treat Regime II as "worth testing" and no-encapsulation calls as untested predictions, not hard exclusions.
-
Structure-dependent
Results are based on a single static structure. Flexibility cannot be measured directly — provide biological context when possible.
-
System-specific
Thresholds are calibrated on one ferritin system. Re-tuning may be needed for other cages or assembly mechanisms.
For full methodological details, assumptions and formulas, see the accompanying manuscript.
Documentation › Examples
Examples
See how enCAGE performs on known proteins.
Run curated examples from our in-house panel or explore results from published cargo–ferritin systems.
At a glance
3
in-house examples5
literature examples3
ferritin systems6
descriptors1
prediction framework1. Try an example
Open in the toolExplore the three proteins used to calibrate enCAGE.
α-Chymotrypsin
Regime IPDB 4CHA
- Compact enzyme
- Near-neutral charge
- Small enough to fit
Net charge (pH 7.4): +4.9
Run this exampleHuman serum albumin
Regime IIPDB 1AO6
- Large, multidomain
- Oversized for cavity
- Likely flexible
Net charge (pH 7.4): −15.1
Run this exampleLysozyme
Regime IIIPDB 1LYZ
- Small, single domain
- Strongly cationic
- Drives off-pathway assembly
Net charge (pH 7.4): +8.7
Run this example2. External validation from the literature
We applied the same three descriptors to independently published cargo–ferritin systems.
Steric compatibility transfers well
Cavity occupancy (volume ratio) predicts fit consistently across ferritin systems.
Electrostatic thresholds are system-specific
Charge remains the key determinant, but the direction and cutoff depend on how the cage assembles.
Descriptors remain informative; labels may not transfer
Equine-calibrated regime labels don't always map directly onto other ferritin systems.
Published examples
| Cargo | Cage | Net charge (pH 7.4) | Reported outcome | Equine-rule regime |
|---|---|---|---|---|
| Cytochrome c1 (chimeric) | AfFtn | +8.4 | Encapsulated | IIIa |
| GFP (+36, supercharged) | AfFtn | +36 | Encapsulated | IIIa |
| GFP | AfFtn | −7.6 | Not encapsulated | Ia |
| GFP (−30, supercharged) | AfFtn | −30 | Not encapsulated | Ia |
| [3Fe–4S] ferredoxin | E. coli Bfr | −8.1 | Encapsulated (4–6 per cage) | Ib |
a AfFtn assembles by co-forming around a sufficiently cationic cargo — the opposite mechanism to equine apoferritin's passive pH-cycling. So a cationic cargo flagged as off-pathway (Regime III) by the equine-calibrated rule can encapsulate cleanly in AfFtn, while a cargo that looks charge-compatible (Regime I) by the same rule can fail to encapsulate if it isn't cationic enough to trigger AfFtn's assembly.
b This acidic cargo is anchored by a cage-specific hydrophobic pocket in Bfr, not by electrostatic complementarity — a reminder that steric fit alone doesn't guarantee the driving mechanism is charge-based.
Visual summary
Charge vs. outcome
(across systems)
+5 cutoff
(AfFtn)
(AfFtn)
(Bfr)
(AfFtn)
(AfFtn)
3. Key takeaways
Regime I reproduces
Compact, charge-compatible proteins encapsulate across systems.
Regime II remains useful
Oversized, flexible proteins are often accommodated — worth testing experimentally.
Charge rules vary
Thresholds and even the direction of electrostatic preference are cage-dependent.
Validation is essential
enCAGE is a heuristic design aid. Always confirm predictions experimentally.
These examples demonstrate how the same descriptors behave across different cargo–ferritin systems. Results may vary for other cages or assembly mechanisms — thresholds are adjustable in the tool.