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FPbase: a community-editable fluorescent protein database

Nature Methods volume 16, pages 277–278 (2019)Cite this article

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To the Editor — The cloning and subsequent engineering of fluorescent proteins (FPs) has revolutionized methods in biological research1,2. There are now thousands of FPs available, each with a different combination of properties that affect its usefulness as a genetic tag in various organelles, cell types, model organisms, and experiments.

The choice of FP can be extremely consequential for the outcome of an experiment3,4, but currently researchers must pore over the large and ever-expanding body of primary literature to make an informed decision. The magnitude of that task incentivizes the reuse of pre-existing, sometimes suboptimal, FPs. A centralized database of FP characteristics would facilitate the dissemination and adoption of new FPs, and encourage the selection of FPs whose strengths are well matched to the researcher’s application. Large-scale aggregation of FP data might additionally facilitate discovery and inferences about FP properties.

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Fig. 1: A portion of an example FPbase fluorescent protein entry.

Code availability

Source code for FPbase is available at https://github.com/tlambert03/FPbase.

Data availability

Although no primary data are presented here, all data collated at https://www.fpbase.org are available for download or upon request.

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Acknowledgements

J. Waters was a strong early advocate of the project, provided invaluable scientific advice throughout the project, and gave feedback on the manuscript. A. Jost provided valuable feedback on data presentation, and G. Campbell helped with data input. N. Shaner and J. Goedhart provided advice on data presentation, and also are members of the FPbase scientific advisory group, along with T. Hughes and A. Palmer. The interactive chart on the FPbase website was modified from an earlier collaboration with K. Thorn. Salary support for T.L. is provided by the Departments of Cell Biology, Systems Biology, and BCMP at Harvard Medical School.

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Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Talley J. Lambert

  2. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    Talley J. Lambert

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  1. Talley J. Lambert
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Correspondence to Talley J. Lambert.

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Integrated supplementary information

Supplementary Figure 1 Advanced Protein Search page.

The advanced search page provides a graphical user interface to search proteins in the database using a combination of filters querying over 20 different protein characteristics. The example shown is a search for all monomeric, constitutively fluorescent (non-photochromic) proteins that have an emission maximum between 500 and 560 and a molecular brightness that is at least 1.5-fold higher than the average brightness of all similarly colored (excitation maximum within 30 nm) proteins in the database published since 2010.

Supplementary Figure 2 Interactive chart of fluorescent protein properties.

The interactive visualization of FP properties plots and compares any two properties across all FPs in the database, or across a subset of proteins filtered using the sliders at the top right of the figure. In this example, the extinction coefficient is plotted as a function of quantum yield, and every circle/square represents a protein in the database. Color corresponds to emission maximum, and numbers correspond to oligomerization. Each object in the chart is interactive: hovering the cursor over an object reveals additional protein properties, and clicking on any item directs the user to the database entry for the respective protein.

Supplementary Figure 3 Example FPbase lineage tree.

Lineage trees integrated throughout the site show mutations and relationships in the directed evolution of fluorescent proteins. This example shows the lineage tree for proteins in the database derived from Entacmaea quadricolor. The input at the top allows the user to search for specific mutations: here the mutations M146T and F174L have been entered, and all proteins possessing both of these mutations are highlighted (amino acid numbering is relative to the root ancestral protein). The graph is interactive: hovering the cursor over an item reveals the mutations used in the generation of that protein from the parental sequence; shown here is the generation of mCardinal from mNeptune2 (S28T/G41Q/S143T).

Supplementary Figure 4 The FPbase spectra viewer.

The FPbase spectra viewer enables the comparison of hundreds of FPs and organic dyes with thousands of filters, light sources, and detectors from many vendors. Shown here is a typical use case: the calculation of emission collection efficiency for multiple FP–emission filter pairs. Spectra are (optionally) shown scaled to excitation efficiency (here, a 561-nm laser), extinction coefficient, and quantum yield. Each table item shows the expected brightness (taking the extinction coefficient, quantum yield, and excitation efficiency into account) and the collection efficiency (the fraction of emission collected by the emission filter) for that FP–filter pair. Pressing the eye icon reveals the overlap integral for that FP–filter pair on the graph.

Supplementary Figure 5 The FPbase FRET calculator.

The FPbase FRET calculator shows spectral overlap and calculates the Förster distance (R0) for any FP pair in the database (provided the spectra and necessary attributes have been entered). The user may change values for the refractive index of the medium (n) or the orientation factor (κ2). The table below shows precalculated Förster distance measurements for all monomeric FP pairs in the database; clicking on the eye icon in the “Show” column displays that FP pair on the graph.

Supplementary Figure 6 Example of an FPbase microscope page.

A form allows users to enter and save the components (filters, light source, camera) in each optical configuration on their microscope(s), and renders a custom spectra viewer, facilitating microscope-specific efficiency calculations for any dye or FP spectrum in the database. Each filter is shown with a link to the respective vendor page, and users may enter notes to be displayed below each optical configuration. A permanent URL allows microscope pages to be shared or embedded elsewhere (such as in a lab or core facility website). The ‘share’ button (below graph to the left (curved arrow symbol)) renders a URL that reconstructs the current settings, for sharing or teaching purposes. Users may also add spectra that are missing from the database.

Supplementary Information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Notes 1–6 and Supplementary Table 1

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Lambert, T.J. FPbase: a community-editable fluorescent protein database. Nat Methods 16, 277–278 (2019). https://doi.org/10.1038/s41592-019-0352-8

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