MicroED Structure of a Protoglobin Reactive Carbene Intermediate

Area of Science:

• Structural Biology
• Biochemistry
• Crystallography

Background:

• Microcrystal electron diffraction (MicroED) is a powerful technique for determining molecular structures.
• While successful for small molecules and peptides, MicroED has had limited application for novel protein structures.
• Advancements in MicroED technology and protein structure prediction are expanding its capabilities.

Purpose of the Study:

• To determine the structure of an engineered Aeropyrum pernix protoglobin (ApePgb) variant using MicroED.
• To utilize an AlphaFold2 model for phasing in the MicroED structure determination.
• To investigate the structural basis for enhanced carbene transfer activity in the ApePgb variant.

Main Methods:

• Microcrystal electron diffraction (MicroED) with advanced technology (higher voltage, low-noise detector).
• Utilized an AlphaFold2 model for phasing.
• Crystallography and structural analysis of the ApePgb variant and its intermediate.

Main Results:

• Determined the first MicroED structure of an Aeropyrum pernix protoglobin (ApePgb) variant.
• Revealed that mutations reorient an alpha helix into a dynamic loop, improving catalytic active site accessibility.
• Trapped and characterized the reactive iron-carbenoid intermediate in the engineered carbene transfer activity.

Conclusions:

• Improved MicroED technology and protein structure prediction models enable the study of previously inaccessible protein structures.
• The determined structure provides insights into how mutations enhance carbene transfer activity.
• The findings highlight the potential of MicroED for investigating complex biological molecules and their mechanisms.