Figure 1: Improvement of Xpo4 export complex crystallization by removal of flexible parts.

(a) Export complexes comprising either full-length or truncated eIF5A were formed with Xpo4 and ZZ-bdNEDD8-tagged RanGTP. After immobilization via tagged Ran, bound proteins were eluted by bdNEDP1 protease⁵⁹, and analysed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie-staining. (b) 180 μg of the export complex was incubated with trypsin (500:1 w/w) for 90 min at 22 °C. The reaction was stopped by 5 mM PMSF and EDTA. Note that the resulting proteolyzed complex remained intact when analysed by size exclusion chromatography (SEC) on a Superdex 200 10/30 column. Experiments with chymotrypsin gave similar results. (c) Illustration of Xpo4 mutants. Protease cleavage sites were identified by mass spectrometry. (d) Overlayed size exclusion chromatograms of the export complexes derived from wild-type Xpo4 or Xpo4 loop deletions. (e) Peak fractions from d were pooled, concentrated and analysed by SDS–PAGE followed by Coomassie-staining. (f) The export complexes from e were incubated with trypsin (1,000:1 w/w) for 1 h at 22 °C and analysed by SDS–PAGE and Coomassie-staining. The ΔLoopN&C Xpo4 variant was trypsin-resistant.