Dataset DOI: 10.5061/dryad.c59zw3rrs

Description of the data and file structure

Dataset title:

Data supporting: A eukaryotic-like phospholipid transfer protein conserved in Asgard archaea

Dataset contents:

This dataset contains one Excel workbook providing the source data associated with the main Figures, Extended Data Figures, and Supplementary Figures of the study.

Excel workbook organization:

Each worksheet is named according to the corresponding figure or panel in the manuscript and contains the numerical source data used to generate that figure.

The workbook also contains the following reference worksheets:

- ProtDomRetriever Results Ref:

Reference results generated using ProtDomRetriever.

- Domain Cavity Size Ref:

Accession numbers and corresponding cavity-size values for the protein domains included in the cavity-size analysis.

- nt_primers_list:

Nucleotide primer sequences used in the study.

Gel images:

Gel images associated with the following Supplementary Figure panels are embedded in their corresponding worksheets of the Excel workbook. Molecular-weight markers are indicated in kDa. Lane identities are as follows:

- Supplementary Fig. 1a
T, total lysate; S, supernatant; P, pellet; FT, flow-through; W1 and W2, wash fractions; MW, molecular-weight marker; E1–E8, successive elution fractions.

- Supplementary Fig. 1b
Lanes correspond to SEC fractions 25, 28 and 30–37; MW, molecular-weight marker.

- Supplementary Fig. 1c
MW, molecular-weight marker; sample lane, purified StarLok1 stock. Lane 3 contains a protein unrelated to this study. Lanes 4–10 contain BSA (bovine serum albumin) standards (125, 250, 500, 750, 1,000, 1,500 and 2,000 µg/mL), loaded at the same volume as the StarLok1 sample.

- Supplementary Fig. 1i
MW, molecular-weight marker; subsequent lanes contain purified 6xHis-StarLok2 and 6xHis-StarLok3. Lanes 4–9 contain BSA (bovine serum albumin) standards (25, 125, 250, 500, 750, 1000 µg/mL), loaded at the same volume as the StarLok1 sample.

- Supplementary Fig. 2c
T, total lysate; S1, supernatant before urea addition; P2, pellet after urea denaturation; S2, supernatant after urea denaturation; FT, flow-through; W, wash fraction; MW, molecular-weight marker; E1–E7, successive elution fractions; Pool, concentrated protein collected before refolding.

- Supplementary Fig. 2d
MW, molecular-weight marker. Sample lanes correspond to 0, 15, 35, 60, 120, and 480 min during refolding in HOAc/NaOAc buffer at pH 4.0 following urea denaturation. Molecular-weight markers are shown on both sides of the gel.

- Supplementary Fig. 4a
Lanes correspond to SEC fractions 21–27; MW, molecular-weight marker, positioned between fractions 22 and 23.

- Supplementary Fig. 4c
Lanes contain refolded StarLok1-containing fractions recovered after elution by affinity chromatography. The dark signal in the second lane after the molecular-weight marker results from oversaturation of the SYPRO Orange stain.

- Supplementary Fig. 4g
MW, molecular-weight marker. For each sucrose-density fraction (Top, 0% sucrose; Medium, 24% sucrose; Bottom, 30% sucrose), samples 1–4 correspond to: 1, no liposome; 2, DOPC liposomes; 3, liposomes containing 5 mol% POPS; 4, liposomes containing 30 mol% POPS.

- Supplementary Fig. 5a
Lane 1, molecular-weight marker; lane 2, purified StarLok1 before Proteinase K treatment; lane 3, StarLok1 after Proteinase K treatment (absence of protein); lanes 4–9, BSA standards used to validate the protein concentration measured before the lipid-transfer assay.

File requirements:

The Excel workbook can be opened using Microsoft Excel or compatible spreadsheet software.

No human participants, personal information, or ethically sensitive data are included in this dataset.

Files and variables

File: Lipp_et_al_Asgard_LTP_Source_Data.xlsx

Description:

This Excel workbook contains the source data associated with the main Figures, Extended Data Figures, and Supplementary Figures of the study. Each worksheet is named according to the corresponding figure or panel. Additional worksheets contain ProtDomRetriever reference results, protein-domain accession numbers with corresponding cavity-size values, and nucleotide primer sequences. 

Variables

Worksheet descriptions

Main Figure 1

Fig1c_spectra
Numerical data underlying the circular dichroism spectra of StarLok1, StarLok2 and StarLok3 shown in Fig. 1c. Mean residue ellipticity is reported as a function of wavelength.

Units: wavelength, nm; mean residue ellipticity (MRE), deg·cm²·dmol⁻¹.

Fig1c_pie_chart
Secondary-structure composition values underlying the pie charts shown in Fig. 1c. Values correspond to the estimated proportions of α-helix, β-strand, turn, and unordered structure obtained from SELCON3 decomposition of the circular dichroism spectra.

Units: secondary-structure composition, %.

Abbreviations: CD, circular dichroism; MRE, mean residue ellipticity.

Main Figure 2

Fig. 2b
Data underlying the comparison of predicted ligand-binding cavity sizes among StarAsg1, StarAsg2 and StarAsg3 homologs identified in Asgard archaea. Protein accession identifiers and corresponding cavity-size values are provided.

Units: cavity volume, ų.
Special value: a value of 0 accompanied by < ND indicates that a cavity size was not determined, as described below under “Missing and special values”.

Fig. 2c
Data underlying the abundance of START-domain classes across Asgard archaeal phyla. Values report the number of identified START domains and the estimated number of species represented in each taxonomic group.

Units: number of identified START domains per species.

Fig. 2d
Data underlying the abundance of START domains in a representative set of archaeal, bacterial and eukaryotic species used for the comparative analyses.

Units: number of identified START domains per species.

Main Figure 3

Fig. 3a
Data underlying the dynamic light scattering analysis of 1 µM StarAsg proteins incubated with liposomes of different lipid compositions. Measurements include the hydrodynamic radius and particle-size polydispersity of liposomes containing DOPC alone or DOPC supplemented with 5, 1,0 or 30 mol% POPS.

Units: hydrodynamic radius, nm; polydispersity, nm; lipid composition, mol%; total lipid concentration

Fig. 3c
Pixel-intensity data underlying the fluorescence profile analysis of StarLok1-GFP and the phosphatidylserine sensor mRFP-C2LACT along the plasma membrane of budding yeast cells.

Units: distance, µm, pixel intensity, arbitrary units;

Abbreviations: DLS, dynamic light scattering; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine; GFP, green fluorescent protein

Main Figure 4

Fig. 4a
Data underlying the total lipid abundance measured for lipids associated with GFP-tagged StarAsg proteins expressed in yeast and analyzed by liquid chromatography–mass spectrometry.

Units: lipid quantity, ng

Fig. 4b
Data underlying the abundance of phospholipid classes associated with StarLok1, StarHod1, StarLok2 and StarLok3.

Units: lipid quantity, ng

Fig. 4d
Numerical data underlying the NBD-lipid transfer kinetic traces for StarLok1 and the corresponding no-protein (1% N-lauroylsarcosine (NLS)) and Proteinase K-inactivated protein controls. The fluorescence signal was normalized to report the absolute amount of NBD-lipid transferred.

Units: time, min; NBD-lipid transferred, µM.

Fig. 4e
Initial lipid-transfer rates were calculated from the initial slopes of the kinetic traces in Fig. 4d, together with the statistical analyses. Each data point represents the initial rate from one separately prepared lipid-transfer assay.

Units: lipid-transfer rate, µM NBD-lipid·min⁻¹.

Abbreviations: LC/MS, liquid chromatography–mass spectrometry; NBD, nitrobenzoxadiazole; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; DHCer, dihydroceramide; ANOVA, analysis of variance; SD, standard deviation; SEM, standard error of the mean.

Extended Data Figure 1

Extended_Data_Fig. 1
Data underlying the distribution of shuttle-like lipid-transfer protein classes across the tree of life. Values report the proportion of analyzed genomes containing each lipid-transfer protein class in Bacteria, Archaea and Eukaryota.

Units: proportion of analyzed genomes, %.
Abbreviation: LTP, lipid-transfer protein.

Extended Data Figure 3

Extended Data Fig. 3a
Cavity-size data underlying the comparison of START-domain phylogenetic classes. Each data point corresponds to the largest predicted ligand-binding cavity identified in one START domain.

Units: cavity volume, ų.

Extended Data Fig. 3b and 3c
Data underlying the t-SNE projections of 805 START-domain structures based on all-versus-all structural similarity. The worksheets contain the two-dimensional t-SNE coordinates, taxonomic/class annotations, and corresponding ligand-binding cavity volumes used to generate Extended Data Fig. 3b and 3c.

Units: cavity volume, ų; t-SNE coordinates, dimensionless.

Abbreviations: START, StAR-related lipid-transfer domain; t-SNE, t-distributed stochastic neighbor embedding.

Reference worksheets

ProtDomRetriever Results Ref
Reference output generated using ProtDomRetriever for the START-domain dataset. The worksheet contains protein identifiers, domain annotations, and positions used to define and extract individual START domains for downstream structural and phylogenetic analyses.

Units: domain positions are amino-acid residue numbers.

Domain Cavity Size Ref
Reference table containing protein/domain accession identifiers and corresponding predicted ligand-binding cavity volumes used in the cavity-size analyses.

Units: cavity volume, ų.

nt_primers_list
Names and nucleotide sequences of oligonucleotide primers used to generate DNA fragments for cloning experiments described in the associated study.

Units: nucleotide sequences are reported in the 5′ to 3′ orientation.

Supplementary Figure worksheets

Supplementary Fig. 1a, Supplementary Fig. 1b, Supplementary Fig. 1c, Supplementary Fig. 1d, Supplementary Fig. 1g, Supplementary Fig. 1h, and Supplementary Fig. 1i
Source data supporting the expression, purification, and biochemical/structural characterization of recombinant StarLok1, StarLok2, and StarLok3 shown in Supplementary Fig. 1. Where applicable, the corresponding gel images are embedded directly in the worksheet.

Supplementary Fig. 2c and Supplementary Fig. 2d
Source data and embedded gel images underlying the corresponding protein purification and biochemical characterization experiments shown in Supplementary Fig. 2.

Supplementary Fig. 4a, Supplementary Fig. 4b, Supplementary Fig. 4c, Supplementary Fig. 4e, Supplementary Fig. 4f, and Supplementary Fig. 4g
Source data supporting the biochemical and biophysical characterization of StarAsg proteins shown in Supplementary Fig. 4, including protein purification, stability and membrane-interaction analyses. Where applicable, gel images are embedded directly in the worksheet.

Supplementary Fig. 5a and Supplementary Fig. 5c
Source data supporting control experiments for the StarLok1-mediated lipid-transfer assay shown in Supplementary Fig. 5. These include the Proteinase K control and the control lipid-transfer measurements. Where applicable, the corresponding gel image is embedded directly in the worksheet.

Common abbreviations: GFP, green fluorescent protein; NBD, nitrobenzoxadiazole; Rh-PE, rhodamine-phosphatidylethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; PS, phosphatidylserine; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Missing and special values:

Blank cells indicate that a value is not available or not applicable.

In the ProtDomRetriever Results Ref worksheet, N/A in Start 3/End 3 or Start 4/End 4 indicates that ProtDomRetriever did not identify a third or fourth domain, respectively.

In the Fig. 2b worksheet, a cavity-size value of 0 indicates that no cavity size could be assigned, either because no cavity was detected or because the cavity could not be reliably defined from the available structure (for example, when it extended beyond the structural model). The adjacent < ND annotation reproduces the notation used in the corresponding figure and denotes a cavity size that was not determined.

Code/software

The dataset consists of a single .xlsx workbook prepared using Microsoft Excel and does not require any specialized software or code to access. The file can be opened using free and open-source spreadsheet software such as LibreOffice Calc, or any other software supporting the Microsoft Excel .xlsx format. No scripts, software packages, or executable files are included in this dataset. The embedded gel images can be viewed directly within the workbook.

Access information

Some reference data used in this dataset were derived from publicly accessible biological databases. Protein accession and annotation information were obtained from UniProt and InterPro. Structural models used for cavity-size analyses were obtained from the AlphaFold Protein Structure Database and, where applicable, the Protein Data Bank (PDB), as described in the associated article. UniProt data are available under CC BY 4.0; InterPro downloadable data under CC0 1.0; AlphaFold Protein Structure Database data under CC BY 4.0. The corresponding accession identifiers are provided in the dataset and/or associated manuscript.