Sub-2 Å Resolution Structure Determination Using Single-Particle Cryo-EM at 200 keV

Abstract

Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination of well-behaved specimens to high-resolution, there nonetheless remains a discrepancy between the resolutions attained for biological specimens and the information limits of modern transmission electron microscopes (TEMs). Instruments operating at 300 kV equipped with DEDs are the current paradigm for high-resolution single-particle cryo-EM, while 200 kV TEMs remain comparatively underutilized for purposes beyond sample screening. Here, we expand upon our prior work and demonstrate that one such 200 kV microscope, the Talos Arctica, equipped with a K2 DED is capable of determining structures of macromolecules to as high as ∼1.7Å resolution. At this resolution, ordered water molecules are readily assigned and holes in aromatic residues can be clearly distinguished in the reconstructions. This work emphasizes the utility of 200 keV for high-resolution single-particle cryo-EM and applications such as structure-based drug design.

2.5 Å-resolution structure of human CDK-activating kinase bound to the clinical inhibitor ICEC0942

“…Clearly, this is not feasible by simply collecting more of the same data, and an increase in either data quality or particle yield is needed. Higher data quality could likely be achieved using improved microscope hardware, as recently described for recordbreaking apo-ferritin structures (16,18), or by increasing the magnification to achieve higher detective quantum efficiency (DQE) at a given spatial frequency (13), with the consequent need to collect more images to obtain the same number of particles. Alternatively, in the light of the extremely low yield of ''good'' particles of $0.2-2% (20,397 and 205,478 particles in the final reconstructions from 9,339,254 particles after initial 2D classification), it will be critical to increase the yield of intact, high-quality particles while producing better orientation distributions.…”

“…In the past few years, cryogenic electron microscopy (cryo-EM) has revolutionized structural biology, allowing structure determination of biological macromolecules that are difficult to crystallize (12). Cryo-EM structures beyond 2-Å resolution have been obtained for a few large or symmetric complexes (13)(14)(15)(16)(17)(18). However, many drug targets are neither large nor symmetric.…”

High-resolution cryo-EM using beam-image shift at 200 keV

“…By taking advantage of microscope aberration correction in RELION-3.1 Zivanov et al , 2020) we were able to improve the resolution of aldolase from 4.9Å to 2.8Å. While previous work demonstrated that aberration refinement allows for resolution improvements for data at both 300 keV (Zivanov et al , 2018) and 200 keV (Zivanov et al , 2020;Wu et al ) , all datasets analyzed were collected on relatively well-aligned instruments. With high-quality starting data, the initial reconstructions prior to aberration correction achieved~3Å (unlike this work which was 4.9Å).…”

“…For example, this was implemented by Henderson and coworkers for the atomic-resolution structure of bacteriorhodopsin from 2D crystals (Henderson et al , 1986) . Since its use 40 years ago, recent advances in single-particle cryo-EM have led to the incorporation of axial beam tilt correction into software packages such as RELION (Herzik et al , 2017;Wu et al ;Zivanov et al , 2020) . The availability of axial beam tilt correction has led to its widespread adoption for cryo-EM structure determination.…”

“…While previous work indicated that short-range beam-image shift could achieve a 3.3Å for the T20S proteasome at 200 keV (Herzik et al , 2017) , this same work required using stage position to obtain a resolution better than 3Å. Recently, using these original datasets of aldolase and T20S datasets, RELION-3.1 now allow higher-order aberrations to be corrected computationally (Zivanov et al , 2020;Wu et al ) . This allowed the resolution of aldolase to improve from 2.5Å to 2.1Å and the T20S proteasome improved from 3.1Å to 2.3Å.…”