Protocols 

“Cryo-EM sample preparation is an arduous and sometimes frustrating process. We have developed new protocols to work with difficult proteins.”

 
 

Gradient fixation

“To stabilize mTORC2, we developed a modified gradient fixation protocol, to be published, building on the original method {link: www.ncbi.nlm.nih.gov/pubmed/18157137} from the Stark laboratory. The modified gradient, illustrated on the left, allows gentle fixation followed by efficient quenching of the cross-linking agent in situ during a single gradient run.

Briefly, a standard gradient mix is supplemented with a small region of both cross-linking agent, in the upper portion of the gradient, and quencher, in the lower. After rotary gradient generation, this results in pulses of each component. Given knowledge of the running parameters of the sample at hand on the gradient, this approach may be adjusted to yield a successively cross-linked and quenched sample.

The resulting sample is suitable for buffer exchange, and can be retrieved by gradient fractionation. More complete details of the protocol are available from our GitHub.”


Carbon floatation

“Adhesion of a protein sample to a thin carbon or graphene film avoiding exposure to air is a frequently used technique in cryo-EM. We have designed a simple block, 3D-printable and available on thingverse {link to thingverse page} (please cite personal communication CHS Aylett), to allow the exchange of sample or buffer under a carbon film, allowing complete control of the adhesion experiment.

A thin carbon film may be floated from an atomically flat mica sheet onto buffer or water, or a layer of graphene dropped onto the surface in solvent, and the sample then subsequently introduced in any buffer, including those containing substances unsuitable for grid preparation, such as sucrose or glycerol, and adhered to the carbon in situ, protected from the air-water interface. The buffer can then be exchanged once more to facilitate the production of high-quality vitreous ice during plunge freezing.

Buffer exchange is facilitated by needle apertures allowing access, whereas the detached mica may be rested at a 45º angle within the well, preventing it from falling, and guiding the experimenter. A minimal sample requirement is allowed due to the low volume of each well (25 uL). Retrieval of the carbon film or graphene layer may be achieved directly onto an applied grid. More complete details are available from our GitHub.”

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