Industrial cryo-EM facility setup and management

Abstract

Cryo-electron microscopy (cryo-EM) has rapidly expanded with the introduction of direct electron detectors, improved image-processing software and automated image acquisition. Its recent adoption by industry, particularly in structure-based drug design, creates new requirements in terms of reliability, reproducibility and throughput. In 2016, Thermo Fisher Scientific (then FEI) partnered with the Medical Research Council Laboratory of Molecular Biology, the University of Cambridge Nanoscience Centre and five pharmaceutical companies [Astex Pharmaceuticals, AstraZeneca, GSK, Sosei Heptares and Union Chimique Belge (UCB)] to form the Cambridge Pharmaceutical Cryo-EM Consortium to share the risks of exploring cryo-EM for early-stage drug discovery. The Consortium expanded with a second Themo Scientific Krios Cryo-EM at the University of Cambridge Department of Materials Science and Metallurgy. Several Consortium members have set up in-house facilities, and a full service cryo-EM facility with Krios and Glacios has been created with the Electron Bio-Imaging Centre for Industry (eBIC for Industry) at Diamond Light Source (DLS), UK. This paper will cover the lessons learned during the setting up of these facilities, including two Consortium Krios microscopes and preparation laboratories, several Glacios microscopes at Consortium member sites, and a Krios and Glacios at eBIC for Industry, regarding site evaluation and selection for high-resolution cryo-EM microscopes, the installation process, scheduling, the operation and maintenance of the microscopes and preparation laboratories, and image processing.

Keywords: cryo-EM; facility management; facility setup; industry.

Figure 1

(a) Schematic of the Consortium IT setup: the Krios 1 and Krios 2 networks are linked by 3× single-mode dark fibres (yellow line) connected to Cisco 9300 switches with 10 Gb modules in the communications rooms for the Cambridge Nanoscience Centre and Department of Materials Science and Metallurgy, respectively. The 450 TB ‘warm’ storage server, Cisco 5515-X ASA hardware firewall and 1 Gbps leased line router are located in the Materials Science communications room (blue lines, OM3 Multimode Fibre, 10 Gbps; grey lines, 1 Gbps ethernet or fibre). The Materials Science communications room is connected to a second Cisco 9300 switch in the Krios 2 room, which is connected to the TEM PC, Support PC and Falcon Offload server. The Nanoscience Centre communications room houses the processing server and both primary and secondary storage servers, as well as the Falcon Offload server (with a direct OM3 connection to Krios 1). The Nanoscience Centre communications room is connected to a second Cisco 9300 switch in the Krios 1 room, which is then connected to the TEM PC, Support PC and the Gatan/K3 server. (b) Physical locations of Krios 1 and Krios 2 on the West Cambridge Site (Google Maps).

Figure 2

For mounting multiple SMB (Samba) connections with different users on the same SMB server to a Windows host, the different users must be mapped to different host names in the hosts file.

Figure 3

The Vitrobot Mk IV ‘blot-force’ steps are 25 µm offsets of the blot pads. The optimal blot force (and blot-pad separation) to obtain a wedge across the grid containing most ice thicknesses is when the blot pads (without filter paper) are just touching or letting a sliver of light through (indicated by the arrow and rectangle). Fine tuning can then be performed by taking full atlases of grids frozen with ±1 or 2 blot-force steps. Increasing the blot force moves the blot pads closer together and therefore the wedge higher (less thick ice areas). Decreasing the blot force moves the blot pads apart and therefore the wedge down (more thick ice areas).

Figure 4

Consortium cryo-cycling procedures. The health-monitoring data from a typical column (a, b) and autoloader (c, d) cryo-cycle with vacuum (a, c) measured by Penning Pirani vacuum gauges (PPcl and PPal) and the corresponding temperatures (b) of the sample ‘holder’ and (d) the autoloader ‘docker’ show that shorter cryo-cycles of 2.5 h for the column and 4.5 h for the autoloader are sufficient.

Figure 5

The pharma Consortium on-the-fly setup consists of pushing data on the fly with Robocopy to the processing server (to an SMB-mounted folder) and simple bash scripts to extract movies (using globstar) and hard link/rename the files to .mrcs (not required for RELION-3). Scheduled RELION is then performed, usually for motion correction and CTF determination to ensure data quality, and also enables a fast start to 2D classification if desired. On-the-fly pipelines are a rapidly developing area, but in most cases some reformatting/transferring/data extraction is needed.