Cryo-EM-Guided Integrative Cross-Linking Mass Spectrometry Pipeline for Resolving Transient Protein–Protein Interaction Interfaces in Spliceosomal B-Complex Assembly

Abstract

Characterizing transient protein–protein interaction (PPI) interfaces within large ribonucleoprotein assemblies remains a fundamental challenge in structural molecular biology. Here, we present an optimized integrative pipeline combining cryo-electron microscopy (cryo-EM) density-guided docking with isotope-labeled cross-linking mass spectrometry (XL-MS) to resolve low-occupancy contact surfaces in the spliceosomal B-complex. By employing NHS-ester and SSD cross-linkers alongside deuterium-labeled peptide standards, we achieved sub-angstrom constraint precision across 14 inter-subunit interfaces involving U2/U5/U6 snRNP components. Integrative scoring against 3.4 Å cryo-EM maps using Bayesian ensemble refinement identified three previously uncharacterized Prp8–SF3B1 contact regions. This methodology offers a robust, reproducible framework for dissecting dynamic PPI networks in macromolecular complexes, with direct implications for splicing-factor-targeted therapeutic strategies.