[3dem] freezing conditions in high salt?

[Rebecca Thompson]

Hi Francesca,

We work with a range of specimens in buffers similar to the ones you describe and get nice ice with good particle contrast, so imagine you won’t have many issues. If your protein is on the small side or has significant disordered regions then the salt concentrations may mean overall image contrast becomes a problem with downstream processing, but with most specimens we work with I don’t worry about 300 mM NaCl too much, if thats what the specimen is happy and stable in.

Parameters seem to vary site to site, machine to machine and sometimes even month to month, but for us, moving the blot arm to ~212- 216 (get this so the grid makes firm flat contact with the filter paper, but grid does not start to bend), blotting 3-5 seconds is the range that gives good results, with the chamber at 8oc and 95 % nominal RH, for R1.2/1.3 holes (larger holes use smaller blot times).  This is blotting so the meniscus directly hits the filter paper (not back blotting). We use Whatman no1 filter paper, and a range of glow discharge instruments but standard glow discharge in air, 30-90 seconds, HT 10. I would say these are the critical parameters, but email me offline and I can provide a full set if it would be useful :)

There are so many parameters you CAN adjust when you are first optimising a new project it can be really tricky to work out what is critical for your system. If its a Leica EM GP thats already in use, asking the previous users what their (successful or unsuccessful!) parameters are will give you a place to start. We also published a review (here<https://journals.iucr.org/d/issues/2018/06/00/ic5104/index.html>) on sample prep which you may find useful.

Hope this helps and good luck!

Best wishes,
Becky

________________________________________________
Rebecca Thompson
Senior cryo-Electron Microscopy Support Scientist/Facility manager
Astbury Biostructure Laboratory
University of Leeds

Email r.f.thompson at leeds.ac.uk<mailto:r.f.thompson at leeds.ac.uk>
Phone 0113 3438957/3438959 (office/Titan Krios control room)
Mobile 07816179813
Location Roger Stevens 5.40
Linkedin   https://uk.linkedin.com/in/1rebeccathompson
Twitter  Bex_16

On 3 Sep 2018, at 10:55, frangube <frangube at gmail.com<mailto:frangube at gmail.com>> wrote:

Dear all,
I was wondering if anyone has experience in obtaining nice, thin ice by freezing a protein in 300mM NaCl (and 20mM Hepes pH 8, no detergent) using a Leica EM GP.
If anyone has suggestions I would really appreciate it!

Thanks in advance.

Francesca

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Dr Francesca Gubellini
Unité Microbiologie Structurale
Institut Pasteur
25 rue du Docteur Roux
75015 Paris
France
Tel: +33 (0)1.40.61.35.31
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