Abstract
Recent advances in single-particle cryo-electron microscopy (cryo-EM) methods have led to the determination of hundreds of high-resolution (< 4.0 Å) macromolecular structures. However, access to high-end instrumentation can be scarce and expensive. The resolution of cryo-EM reconstructions is fundamentally limited by the Nyquist frequency, which is half the sampling frequency of the detector and depends upon the magnification used. In principle, super-resolution imaging should enable reconstructions to surpass the physical Nyquist limit by increasing sampling frequency, yet there are no reports of reconstructions that do so. Here we report the use of super-resolution imaging with the K3 direct electron detector to produce super-resolution cryo-EM reconstructions significantly surpassing the physical Nyquist limit. We demonstrate that super-resolution imaging can be used to capture high-resolution information at lower magnifications, facilitating the imaging of thousands of particles per micrograph and maximizing the use of limited instrument time. Remarkably, using this approach we produced a 3.7 Å reconstruction of jack bean urease with particles selected from a single micrograph.