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MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel

Michael W Martynowycz1,2, Farha Khan3, Johan Hattne1,2

  • 1Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095.

Proceedings of the National Academy of Sciences of the United States of America
|December 9, 2020
PubMed

Insights

Researchers determined the structure of a murine voltage-dependent anion channel (VDAC) mutant using microcrystal electron diffraction (MicroED). This method successfully solved the structure of VDAC microcrystals grown in viscous media, overcoming limitations of X-ray crystallography.

Area of Science:

  • Structural Biology
  • Membrane Protein Research
  • Electron Crystallography

Background:

  • The voltage-dependent anion channel (VDAC) is a crucial membrane protein involved in cellular metabolism and transport.
  • Obtaining high-resolution structures of membrane proteins, especially in their native-like lipidic environments, remains a significant challenge for conventional structural biology techniques.
  • Microcrystal electron diffraction (MicroED) has emerged as a powerful alternative for determining structures from small or difficult-to-crystallize samples.

Purpose of the Study:

  • To determine the high-resolution structure of a specific mutant of the murine voltage-dependent anion channel (VDAC).
  • To demonstrate the utility of microcrystal electron diffraction (MicroED) coupled with focused-ion beam (FIB) milling for structural analysis of membrane proteins.
  • To investigate the role of lipids in the crystal packing and stability of VDAC.

Main Methods:

  • Growing microcrystals of a VDAC mutant in a viscous bicelle suspension.
  • Identifying suitable thin, plate-like crystals using scanning electron microscopy (SEM).
  • Preparing crystal lamellae using focused-ion beam (FIB) milling.
  • Collecting and merging MicroED data from multiple crystal lamellae for structure determination.

Main Results:

  • The refined structure of the VDAC mutant was obtained using MicroED.
  • Unmodeled densities between VDAC monomers suggested the presence of lipids, potentially mediating inter-protein contacts within the crystal.
  • The study successfully demonstrated the integration of FIB milling and MicroED for membrane protein structure determination.

Conclusions:

  • Microcrystal electron diffraction (MicroED), combined with FIB milling, is an effective strategy for determining the structures of membrane proteins that are intractable by X-ray crystallography.
  • The developed approach enables structural studies of proteins crystallized in lipidic environments, providing insights into their native-like states.
  • This work presents a novel structure of a VDAC mutant, highlighting the potential of MicroED for advancing membrane protein structural biology.
Keywords:
FIB/SEMMicroEDbicelle crystallizationcryoEMmicrocrystal electron diffraction

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