Reference-Based Multi-Lattice Indexing Method Integrating Prior Information in Free-Electron Laser Protein Crystallography

X-ray free-electron lasers (XFELs) have revolutionized structural biology by enabling “diffraction-before-destruction” and capturing the ultrafast dynamics of life. However, the intrinsic sparsity and noise of XFEL diffraction snapshots, often complicated by multi-lattice overlaps, create a formidable computational bottleneck that limits data utilization and structural fidelity. Here, we present MCDPS-SFX, a robust indexing framework based on a reference-based, whole-pattern matching principle integrated with parallelized iterative refinement. By exhaustively sampling orientation space and progressively rejecting outliers, MCDPS-SFX significantly outperforms legacy algorithms—more than doubling crystal yields in heterogeneous datasets (e.g., 21,807 vs. 8792 for MOSFLM)—and achieves highly competitive yields comparable to state-of-the-art indexers, such as extracting over 90,000 lattices in the lysozyme benchmark. We demonstrate its efficacy on standard benchmarks and technically demanding G-protein-coupled receptor (GPCR) systems, including the rhodopsin–arrestin complex and the glucagon receptor. MCDPS-SFX consistently produces high-quality data statistics, enabling the high-resolution visualization of functionally critical, flexible regions such as phosphorylated receptor tails. Our results provide a powerful tool for enhancing the scientific output of XFEL experiments, offering a robust alternative for maximizing information recovery from weakly diffracting or overlapping crystalline samples.