Automated vitrification of cryo-EM samples with controllable sample thickness using suction and real-time optical inspection.

The speed and efficiency of data collection and image processing in cryo-electron microscopy have increased over the last decade. However, cryo specimen preparation techniques have lagged and faster, more reproducible specimen preparation devices are needed. Here, we present a vitrification device with highly automated sample handling, requiring only limited user interaction. Moreover, the device allows inspection of thin films using light microscopy, since the excess liquid is removed through suction by tubes, not blotting paper. In combination with dew-point control, this enables thin film preparation in a controlled and reproducible manner. The advantage is that the quality of the prepared cryo specimen is characterized before electron microscopy data acquisition. The practicality and performance of the device are illustrated with experimental results obtained by vitrification of protein suspensions, lipid vesicles, bacterial and human cells, followed by imaged using single particle analysis, cryo-electron tomography, and cryo correlated light and electron microscopy.

Stabilizing the closed SARS-CoV-2 spike trimer.

The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify regions critical for stability: the heptad repeat region 1, the SD1 domain and position 614 in SD2. We combine a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.