In-Cell Protein Crystallization via a Locally Flexible 24-mer Assembly Precursor

In-cell protein crystallization (ICPC) is a process that creates well-ordered protein crystals within living cells, yet the methods proteins utilize to achieve long-range crystalline order within the cellular setting are not fully understood. This study identifies the assembly process of CipB, a protein that forms crystals in Photorhabdus luminescens.

Upon crystallization within cells, CipB crystals break down in mildly acidic conditions, revealing a dominant 24-mer species, which suggests that a specific 24-mer assembly precursor is central to in-cell crystal formation.

Structural studies of the recrystallized CipB protein show that the same 24-mer arrangement forms a body-centered cubic lattice, mirroring the lattice structure of the in-cell crystals. Cryo-electron microscopy (cryo-EM) and molecular dynamics simulations reveal that the 24-mer precursor maintains its overall structure while allowing for local flexibility at the N-terminal and surface-loop regions.

Additionally, mutation studies establish a connection between the N-terminal region and the formation of the 24-mer precursor, and between surface residues and the organization of these precursors into the lattice assembly.

These findings support a stepwise crystallization model, where the N-terminal flexibility plays a crucial role in the formation of the assembly-competent 24-mer precursor, while specific hydrophobic surface interactions subsequently arrange these precursors into a long-range-ordered crystalline lattice.