Conformational bias imposed by source microseeds results in structural ambiguity

Area of Science:

• Cellular signaling
• Molecular biology
• Structural biology

Background:

• The p38 MAP kinase pathway is crucial for cellular responses to environmental stress.
• Alternative activation pathways exist, including a T cell-specific pathway involving TCR activation and Tyr323 phosphorylation.
• Understanding these pathways is vital for immunology and drug development.

Purpose of the Study:

• To investigate structural changes in p38 MAP kinase during T cell-specific activation.
• To evaluate the impact of specific mutations on p38 conformation and autoactivation.
• To assess the influence of cross streak-seeding techniques on protein crystallization outcomes.

Main Methods:

• Site-directed mutagenesis to create active and inactive p38 mutants at Tyr323.
• Cross streak-seeding techniques to promote crystal growth.
• X-ray crystallography to determine the structures of wild-type and mutant p38.

Main Results:

• Mutants exhibited varying crystal characteristics, with some adopting the wild-type crystal form (A) and others forming a new form (B).
• The Y323T mutant crystallized in both forms, revealing distinct molecular conformations dictated by the seeding crystals.
• Cross streak-seeding can influence the observed molecular structure, potentially introducing bias.

Conclusions:

• The source seeds used in cross streak-seeding can impose conformational bias on crystallization outcomes.
• This phenomenon may lead to crystal structures that do not accurately represent the molecule's authentic conformation.
• Careful consideration of seeding methods is necessary for accurate structural determination in crystallography.