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The specimen preparation process is a key determinant in the success of any cryo electron microscopy (cryoEM) structural study and until recently had remained largely unchanged from the initial designs of Jacques Dubochet and others in the 1980s. The process has transformed structural biology, but it is largely manual and can require extensive optimisation for each protein sample. The chameleon instrument with its self-wicking grids and fast-plunge freezing represents a shift towards a robust, automated, and highly controllable future for specimen preparation. However, these new technologies require new workflows and an understanding of their limitations and strengths. As early adopters of the chameleon technology, we report on our experiences and lessons learned through case studies. We use these to make recommendations for the benefit of future users of the chameleon system and the field of cryoEM specimen preparation generally.

Urease is a nickel (Ni) enzyme that is essential for the colonization of Helicobacter pylori in the human stomach. To solve the problem of delivering the toxic Ni ion to the active site without diffusing into the cytoplasm, cells have evolved metal carrier proteins, or metallochaperones, to deliver the toxic ions to specific protein complexes. Ni delivery requires urease to form an activation complex with the urease accessory proteins UreFD and UreG. Here, we determined the cryo–electron microscopy structures of H. pylori UreFD/urease and Klebsiella pneumoniae UreD/urease complexes at 2.3- and 2.7-angstrom resolutions, respectively. Combining structural, mutagenesis, and biochemical studies, we show that the formation of the activation complex opens a 100-angstrom-long tunnel, where the Ni ion is delivered through UreFD to the active site of urease.

Cryogenic electron microscopy (cryo-EM) is a leading technique for determination of the structure of proteins and protein complexes. However, preparation and freezing of cryo-EM grids is often an iterative process and generation of grids that produce high quality data sets is difficult if not impossible for some proteins, particularly those that display pathological preferred orientation or air/water interface denaturation [1][2][3][4]. Conventional plunging techniques, which involve contacting one or both sides of the grid with a blotting paper, generate thousands of air/water interface interactions per protein during the plunging process in addition to denaturation effects from the blotting paper itself [5][6][7]. In recent years, an explosion of next-generation cryo-EM sample preparation instruments have been generated or manufactured, ranging from in-house models such as the Back-it-up [8] to commercially available models such as the chameleon (developed by SPT Labtech and based on the Spotiton developed by Carragher et al.) [9][10][11]. The chameleon utilizes a piezoelectric dispenser and self-wicking nanowire grids with a tunable dispense-to-plunge time based on strength and duration of glow discharge; this allows for a blot-free device that can accommodate a range (2500 -54 ms) of dispense-to-plunge times.

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