Fig. 2: In vitro GEF assays and negative stain EM analysis reveal that the reconstituted core Rab3GAP complex retains GEF activity towards Rab18 and adopts only one main conformation.

a Schematic of Rab3GAP1 with GAP domain annotated, and Rab3GAP2 domain 1 and Rab3GAP2 domain 2 connected by a flexible linker. b Analytical gel filtration elution profile of core Rab3GAP on an ENrich™ SEC 650 column and representative SDS-PAGE gel of the reconstituted complex stained with Coomassie Blue. Analytical gel filtration and SDS-PAGE gels were performed in biological triplicate (n = 3). c GEF assays on core Rab3GAP. Nucleotide exchange was detected by measuring fluorescent decrease in reactions containing 0 nM GEF (Mock) or 300 nM GEF with 4 µM Mant-GDP loaded Rab18 and 100 µM GTPγS. Data are presented as mean with error bars showing SEM for assays performed in technical triplicate (n = 3). d Representative 2D class averages comparing the general architecture of core Rab3GAP to full-length Rab3GAP. Schematic showing only a single conformation for core Rab3GAP compared to the open and closed conformation of full-length Rab3GAP, with Rab3GAP1 in blue and Rab3GAP2 in pink. Source data are provided as a Source Data file.