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If you cannot see it, is it still there? – Ensemble refinement of invisible protein segments

Summary

Scientists studying protein structures using X-ray crystallography often face a problem: flexible parts of proteins don’t show up clearly in their images and get left out of molecular models. This paper demonstrates a new technique called ensemble refinement that can visualize these ‘invisible’ flexible regions by creating multiple model versions simultaneously. Using a mushroom enzyme that produces psilocybin as an example, researchers show that this approach reveals how protein parts actually move and change shape, providing better understanding of how proteins function.

Background

X-ray crystallography determines protein structures through electron density mapping, but flexible regions of proteins often remain invisible in electron density maps and are omitted from structural models. This creates an incomplete representation of molecular structures despite these regions being covalently present in the crystal.

Objective

To demonstrate that ensemble refinement (ER) can visualize and analyze the conformational landscape of ‘invisible’ protein segments, particularly flexible regions with functional roles. The study uses the fungal methyltransferase PsiM as an example to illustrate how ER of multiple crystal forms can reveal substrate recognition loop dynamics.

Results

Ensemble refinement revealed that PsiM’s substrate recognition loop (SRL), appearing rigid in high-resolution structures, is genuinely flexible and explores multiple conformations in the absence of substrate. Different crystal forms showed context-dependent conformational spaces, with the SRL potentially functioning as a dynamic ‘flap’ for substrate binding and release.

Conclusion

Ensemble refinement provides effective visualization of protein flexibility and molecular plasticity, particularly when applied to multiple crystal forms. This approach better represents the dynamic nature of proteins compared to static models and offers insights into functional aspects of molecular flexibility.
  • Published in:Journal of Applied Crystallography,
  • Study Type:Methods Development,
  • Source: 10.1107/S160057672500130X, 40170967
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