2-592~, SdeC~222-592~, SidE~219-591~, DupA, DupB, lpg1496~297-Cterm~, lpg2523~492-Cterm~ and DupA~4-345~ (crystallized construct) were cloned into pParallelGST2 vector ([@bib61]). Full length SdeA is cloned into pGEX-6P-1 vector. T7 express *E.coli* competent cells (NEB) were transformed with plasmids and grown in LB medium to an OD~600~ of 0.6-0.8 at 37°C. Protein expression was induced by addition of 0.5 mM IPTG (isopropyl ~D~-thiogalactopyranoside) and cells were further grown overnight at 18°C and harvested. The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl pH 7.5 and 150 mM NaCl, 1 mM TCEP) and lysed by sonication and centrifuged at 13,000 rpm to clarify the supernatant. The supernatant was incubated 1 h with glutathione-*S*-Sepharose pre-equilibrated with washing buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl) and non-specific proteins were cleared with washing. GST-proteins were eluted with elution buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 15 mM reduced glutathione) and buffer exchanged to storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl). For DupA~4-345~ WT, H67A or H67A/H189N, instead of using elution buffer, glutathione beads were incubated with sfGFP-TEV protease ([@bib72]) overnight at 4°C. Eluted proteins were further purified by anion exchange chromatography on HitrapQ (GE Healthcare) and fractions contacting samples were loaded onto size exclusion column (Superdex 75 16/60, GE Healthcare) pre-equilibrated with 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM TCEP. Proteins were concentrated to 10 mg/mL and stored for crystallization. PR-ubiquitinated Rtn4 peptide were synthesized by incubation with GST-SdeA~FL~, NAD, Ub in the reaction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) at 37°C for 2 h. To effectively modify the peptide, Ub were iteratively added to reaction mixture every 30 min ([@bib62]). PR-ubiquitinated peptide were further purified with Ni-NTA agarose and size exclusion chromatography (Superdex 30 prep grade 16/60, GE Healthcare).

### Crystallization {#se