(a) Composite volume of locally refined regions determined using cryogenic electron microscopy for ISG65 bound to human C3b. ISG65 is coloured in different shades of blue and green, as indicated in …
Steps highlighted in blue were performed in SIMPLE, steps in green were performed in CryoSPARC, and steps in orange were performed in RELION.
(a) The ISG65-C3b model shown in transparent cryo-EM density. The top panel shows the interface between ISG65 and the TED domain (orange), with bottom panel showing the interface between loop L2 of …
Surface plasmon resonance data.
Two experimental replicates of the data are shown in Figure 2c. Twofold serial dilutions from a concentration range of 5000–312.5 nM. Data shown here is derived from two biological replicates of …
Surface plasmon resonance data.
Surface plasmon resonance data.
(a) The structure of the ISG65-C3b complex (without ISG65) docked into the electron microscopy-derived volumes obtained for the ISG65-C3b complex (left) and C3b alone (right). (b) Composite models …
Figure 3c – raw gel 1 annotated.
Figure 3c – raw gel 1.
Figure 3c – raw gel 2 annotated.
Figure 3c – raw gel 2.
Figure 3d – raw gel annotated.
Figure 3d – raw gel.
Figure 3e – raw gel annotated.
Figure 3e – raw gel.
Composite models obtained by docking the C3b-ISG65 structure onto those of C3b/d bound to factor H CCP19-20 (3OXU) (Morgan et al., 2011), CRIg (2ICF) (Wiesmann et al., 2006), CR1 CCP15-17 (5FO9) (For…
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Trypanosoma brucei brucei) | ISG65G gene | NCBI BioProject Accession: PRJEB46985 | UniProt: A0A8J9S0Z8 | |
Gene (Homo sapiens) | CFB gene | NCBI GenBank accession: AF019413.1 | UniProt: P00751 | |
Gene (H. sapiens) | CFD gene | NCBI GenBank accession: CH471139.2 | UniProt: P00746 | |
Gene (H. sapiens) | C3 gene | NCBI GenBank accession: AY513239.1 | UniProt: P01024 | |
Sequence-based reagent | Primers | This paper | See list of primers in the Appendeix. Primers were synthesised by Sigma. | |
Recombinant DNA reagent | pHL-SEC vector backbone | https://doi.org/10.1107/S0907444906029799; Aricescu et al., 2006 | ||
Cell line (H. sapiens) | HEK293F | Gibco | R79007 | |
Biological sample (H. sapiens) | Human serum | NHSBT non-clinical issue | ||
Software | SIMPLE v3 | https://github.com/hael/SIMPLE/releases; Elmlund et al., 2020 | ||
Software | CryoSPARC v3 | https://cryosparc.com/; Structura Biotechnology Inc, 2020 | ||
Software | TOPAZ v0.2.4 | https://github.com/tbepler/topaz; Bepler and Noble, 2020 | ||
Software | RELION v3.1 | https://relion.readthedocs.io/en/release-3.1/index.html; RELION developer team, 2020 | ||
Software | DeepEMhancer | https://github.com/rsanchezgarc/deepEMhancer; Sanchez Garcia, 2022 | ||
Software | AlphaFold2 | https://github.com/google-deepmind/alphafold; AlphaFold Team, 2021 | ||
Software | ISOLDE v1.0 | https://github.com/tristanic/isolde; Croll, 2019 | ||
Software | COOT v0.9.8.3 | https://github.com/pemsley/coot; Emsley, 2022 | ||
Software | PHENIX v1.20.1 | https://phenix-online.org; Phenix Development Group, 2022 | ||
Software | ChimeraX v1.6 | https://www.cgl.ucsf.edu/chimerax/; UCSF Resource for Biocomputing, Visualization, and Informatics, 2023 | ||
Software | BIAevaluation v1.0 | Biacore, Cytiva, Marlborough, MA, USA | ||
Software | Fiji | https://imagej.net/software/fiji/ |
Supplementary Table 1: Cryo-EM data collection and model building statistics.
Supplementary Table 2: Kinetic parameters of ISG65 variants binding to biotinylated C3b and C3d, as measured by surface plasmon resonance.
Average values and standard deviation from three experimental repeats are reported.
Supplementary Table 3: Mass spectrometry analysis of ISG65-C3b conjugates observed in Figure 3.
The high molecular weight band observed in convertase assay in the presence of ISG65 was excised and analysed by mass spectrometry. The three most abundant proteins identified are presented.
Primers.