Resolving smaller molecules with cryo-EM

At this year’s annual meeting of the American Crystallographic Association in New Orleans, Maryam Khoshouei of the Max Planck Institute of Biochemistry (MPIB) described how she and her collaborators used a novel technique to obtain a 3.2-Å-resolution structure of human hemoglobin (see accompanying figure). The feat is significant because, at 64 kDa, the protein complex is smaller than the 100 kDa lower limit that had previously prevailed when using cryoelectron microscopy (cryo-EM) to determine the structure of protein complexes.

In EM, contrast arises from differences in phase between scattered and unscattered electrons. (See the article by Bob Glaeser, Physics Today, January 2008, page 48 .) To create that difference, microscopists either defocus their beams or insert an ultrathin phase plate with a micron-sized hole at the focus; scattered electrons that pass through the plate acquire a phase shift of π/2 with respect to unscattered electrons that fly straight through the hole. Neither approach is ideal. Defocus leads to the diminution or even loss of low spatial frequencies, whereas holed phase plates are challenging to align and keep stable.

To mitigate either of those problems, the MPIB team developed a phase plate that lacks a central hole. Made from a 10-nm-thick sliver of amorphous carbon, the phase plate imposes a phase difference between the central, unscattered beam and its scattered periphery through the spontaneous interaction of the beam’s electrons and the plate’s atoms. At first, the MPIB researchers thought they needed to use their phase plate with a tightly focused—and therefore painstakingly aligned—beam. They later discovered that they could retain most of the setup’s performance with a slightly defocused beam.

In single-particle cryo-EM, molecules in aqueous solution are abruptly frozen and imaged in whatever conformational state they happen to be. Not only does that step dispense with the need to make a crystal, it also makes it possible to analyze the range of states that enzymes and other proteins adopt in vivo. Recent advances in electron detectors have greatly reduced the time it takes to determine structures with cryo-EM. (See Physics Today, August 2016, page 13 .) The new phase plate will likely help to bring cryo-EM even closer to joining x-ray crystallography as a routine method for high-resolution protein-structure determination. (M. Khoshouei et al., Nat. Commun. 8, 16099, 2017, doi:10.1038/ncomms16099 .)