Extended Data Fig. 2. Synthesis of ABPs to capture TS1 and TS2.

a, Strategy to generate an ABP to visualize TS1 (TS1 ABP). The goal was to generate an ABP with a warhead between the catalytic cysteine of UBE2L3 and the C terminus of ubiquitin that would react with ARIH1 only when assembled with a neddylated CRL. An intein-based semisynthesis route was used to couple Ub(1–75)–MESNa and (E)-3-[2-(bromomethyl)-1,3-dioxolan-2-yl]prop-2-en-1-amine (BmDPA) to yield a cyclic ketal-protected ubiquitin species. Acidic deprotection of the cyclic ketal yields a reactive ubiquitin species⁶⁵, which when conjugated to a single-cysteine-containing version of UBE2L3 produces an ABP with a Michael acceptor between the C terminus of ubiquitin and the active site of UBE2L3. b, Quality controls comparing predicted masses for Ub–MESNa, ABP precursor and TS1 ABP entities with measurements obtained by electrospray ionization–time-of-flight mass spectrometry. c, SDS–PAGE gel confirming TS1 ABP reaction depends on the catalytic cysteine of ARIH1 (C>S refers to serine replacement). Gel image is representative of independent technical replicates (n = 2). d, SDS–PAGE gel demonstrating TS1 ABP reaction with ARIH1 depends on neddylated CUL1–RBX1 and ARIH1 residues required for ubiquitylating client substrates bound to an F-box protein⁴. Gel image is representative of independent technical replicates (n = 2). e, Because structural biology is an empirical endeavour, various TS2 ABP approaches were tested to identify a strategy yielding high-quality electron microscopy data visualizing TS2. The concept was to place a warhead between a substrate and the C terminus of ubiquitin, to generate an ABP that would react only with ARIH1 super-assembled with the SCF containing the cognate F-box protein of the substrate. The fully synthetic TS2 ABP alternative (left) displayed reactivity and specificity matching the native reaction when assembled with cyclin E or p27 phosphopeptide substrate mimics, as shown by SDS–PAGE gel (right). f, In parallel, we tested a semisynthetic strategy, which led to high-resolution cryo-EM structures, and thus complexes generated with this strategy are referred to as TS2 throughout the article. Ub–MESNa and a substrate phosphopeptide with an N-terminal cysteine placed to mimic the acceptor site were fused via native chemical ligation and the free cysteine was converted to dehydroalanine. g, SDS–PAGE showing TS2 p27 ABP reaction with ARIH1 requires all elements needed for native TS2, or use of a mutant version of ARIH1 (ARIH1(F430A/E431A/E503A)) bypassing the need for NEDD8 for this reaction. Gel image is representative of independent technical replicates (n = 2). h, SDS–PAGE testing specificity of TS2 ABPs for cognate F-box proteins. Phosphorylated cyclin E and p27 are substrates of SCF^FBXW7 and SCF^SKP2, respectively. All experiments with SCF^SKP2 also contained the essential protein partner CKSHS1 unless otherwise indicated. Gel image is representative of independent technical replicates (n = 2).