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Related Concept Videos

Cryo-electron Microscopy01:28

Cryo-electron Microscopy

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Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...
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A fiducial-assisted strategy compatible with resolving small MFS transporter structures in multiple conformations

Pujun Xie1, Yan Li2, Gaëlle Lamon1

  • 1Department of Chemistry, New York University, New York, NY, USA.

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This study introduces a novel cryo-electron microscopy (cryo-EM) strategy using a rigid fiducial marker (BRIL domain) to determine structures of challenging membrane transporters. This method successfully resolved multiple conformations of the NorA transporter, advancing structural biology.

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Area of Science:

  • Structural Biology
  • Biophysics
  • Molecular Biology

Background:

  • Cryo-electron microscopy (cryo-EM) has advanced structural biology, but challenges remain for small membrane proteins (<40 kDa).
  • Determining multiple protein conformations from a single sample is difficult, especially for transporters and G-protein coupled receptors.
  • Lack of distinguishable features hinders image alignment and structure determination for these proteins.

Purpose of the Study:

  • To develop a strategy for determining high-resolution cryo-EM structures of membrane transporters near the size threshold.
  • To enable the characterization of multiple conformational states within a single sample.
  • To validate a novel fusion construct approach for cryo-EM studies of Major Facilitator Superfamily (MFS) transporters.

Main Methods:

  • Fusion of the target transporter's C-terminus with the BRIL domain using a poly-alanine linker.
  • Utilizing AlphaFold2 predictions to guide construct design and minimize steric clash.
  • Employing a BRIL-specific Fab fragment for enhanced particle orientation and image alignment.
  • Applying cryo-EM to elucidate structures of the Staphylococcus aureus NorA transporter and three other MFS transporters.

Main Results:

  • Successfully determined four cryo-EM structures of the 42 kDa NorA transporter.
  • Resolved three distinct conformations (inward-open, inward-occluded, occluded) of NorA from a single sample.
  • Validated the fusion strategy for three additional MFS transporters (GlpT, Bmr, Blt), confirming the utility of a rigid linker.
  • Demonstrated that AlphaFold2 predictions facilitated efficient construct design.

Conclusions:

  • The BRIL fusion strategy is effective for cryo-EM structure determination of small membrane transporters, including MFS family members.
  • This method facilitates the capture and analysis of diverse protein conformations from a single experiment.
  • The approach significantly reduces guesswork and screening, making cryo-EM more accessible for studying membrane protein dynamics.