New image analysis method breaks through cryo-EM resolution limits

A research group led by Project Assistant Professor Raymond N. Burton Smith and Project Professor Kazuyoshi Murata of the Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, and the National Institute for Physiological Sciences (NIPS), developed PASR (Post-Acquisition Super Resolution), a new image analysis technology that can restore high-resolution information beyond conventional physical resolution limits from data acquired by cryo-electron microscopy (cryo-EM). The technique enables researchers to extract more detailed structural information from existing data without additional microscope measurements and is expected to improve the efficiency and accuracy of cryo-EM analysis in structural biology and drug discovery research. The findings were published online in IUCrJ.

Cryo-EM has rapidly developed as a technology capable of determining the three-dimensional structures of proteins, macromolecular complexes, virus particles, and other biological entities at atomic resolution. In particular, advances in single-particle analysis (SPA) have enabled the analysis of membrane proteins and macromolecular complexes that are difficult to crystallize, making cryo-EM an important tool in life science and drug discovery research.

However, cryo-EM is subject to a physical resolution limit known as the Nyquist limit, the theoretical limit of detail that can be recorded in digital images. Determined by the camera's pixel size, this limit prevents finer structural information from being recorded accurately. Although higher resolution can be achieved by reacquiring data at higher magnifications, high-magnification imaging reduces the number of particles captured in each image, significantly increasing acquisition time and data storage requirements.

The PASR method developed by the research group subdivides each pixel in previously acquired movie data computationally and uses minute positional shifts between frames during motion correction to recover high-frequency information that could not previously be obtained.

The team analyzed datasets of apoferritin, jack bean urease, adeno-associated virus (AAV), nodavirus, and the giant virus Melbournevirus, confirming resolution improvements beyond the physical Nyquist limit even from standard counting-mode data. In addition, PASR can be used with widely adopted cryo-EM analysis software packages such as RELION and CryoSPARC and does not require a specialized analysis environment. Furthermore, when combined with super-resolution imaging data, the method successfully achieved reconstructions exceeding the conventional super-resolution Nyquist limit.

Because PASR enables high-resolution analysis while imaging large numbers of particles at low magnifications, it is expected to improve data acquisition efficiency, reduce microscope usage time, and lower data storage requirements.

The research group plans to integrate PASR into cryo-EM analysis pipelines and apply the technology to real-time processing in the future.

Journal Information
Publication: IUCrJ
Title: Post-acquisition super resolution for cryo-electron microscopy
DOI: 10.1107/S2052252526005348