[3dem] [ext] Hexamer dissociate while freezing Cryo-EM grids

Fri Jun 5 02:54:44 PDT 2020

Hi Umar,

You will see from the many responses that you are not alone in experiencing this problem of a specimen looking significantly different in negative stain vs cryoEM! Unfortunately, at present there is no real ‘silver bullet’ to tackle this, and the only way forward is to test a whole load of different variables until you find the thing that works for your protein. There have been some excellent suggestions so far, but wanted to expand on these;

 (Caveat- only start doing these if you are confident in the quality of your prep-, you say neg stain looks nice and I expect you have biochemistry to back up (SEC (MALLS), native gels, native MS, CD, functional assays etc).

1) The first thing to check is that your particle is definitely dissociating and that you don't have intact particles. There could be other reasons why you aren’t seeing particles in the holes of your grids. The particles could strongly partition to the carbon support film, in these cases it can sometimes be hard to distinguish particles from the amorphous carbon. If you particles are there, but on the carbon, try altering glow discharge/plasma treatment, change to a different support material (like ultrAufoils), or investigate the use of a continuous film (GO or continuous carbon, depending on the size of your particle). As Ed pointed out, a second reason could be that your ice is so thin that you are physically excluding the particles from the holes, in which are looking for thicker ice will confirm this, but then smaller hole diameters may help to achieve more uniform thickness ice without the thinning in the middle that leads to particle exclusion from the holes. This is most common with larger specimens such as viruses.

2) If you are sure the particle is dissociating, as others have pointed out the AWI is potentially to blame. There is a lot of excellent literature on why this is the case (including the excellent paper Davide pointed out) but heres a quick summary of things you can try, in no particular order;

A) Move the particle away from the AWI by sequestering it at the support water interface. Includes easy-to-try options like continuous carbon (if you have a particle where you have sufficient contrast to still align with the additional noise from the carbon), graphene oxide (or other engineered graphene supports- https://urldefense.com/v3/__https://www.pnas.org/content/116/24/11718__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxHSc3BrSg$ ), or ‘affinity grids’ including the srreptavadin monolayers that were mentioned, although these might involve some additional work with your protein.

B) Change the properties of the AWI by using a surfactant- unfortunately a huge range exist and it seems that one detergent or concentration isn’t the magic formula that works for all proteins, so there may be a lot of variables for you to try here. Common ones to try are DDM, LMNG, CHAPSO, NP40)(stick to sub CMC concentrations for detergents).

C) Try a different device. The shear forces induced by filter paper based blotting might be your culprit (https://urldefense.com/v3/__https://doi.org/10.1016/j.bpj.2019.12.017__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxFz03UNHQ$ ). Spotiton/Chameleon/Back-It-Up/Shake-it-off, CryoWriter and time resolved cryoEM devices all avoid using filter paper. But in terms of interactions with the AWI , these occur within milliseconds- so even with the fastest of these you wont out run AWI interactions and the potential effects (see the preprint in D)

D) If you can, apply a higher concentration. If the protein itself is denaturing at the AWI, it will act as a surfactant which may prevent further denaturation. Saturating the AWI may also have positive effects on the angular distribution of particles that remain in the bulk of the thin film (away from the AWI)- https://urldefense.com/v3/__https://doi.org/10.1101/2020.05.14.095372__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxHibkKL_g$ .

E) Approach the problem from a biochemical perspective and crosslink the sample.

We also published a bit of a review about different variables that can be changed to alter particle distribution- https://urldefense.com/v3/__https://journals.iucr.org/d/issues/2018/06/00/ic5104/index.html__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxHoNQuhLQ$ 

Sorry this is a bit of a long email, but hope it is useful. 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 3433384/3438957 (office/Titan Krios control room)
Mobile 07816179813
Location Roger Stevens 5.40
Linkedin   https://urldefense.com/v3/__https://uk.linkedin.com/in/1rebeccathompson__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxE7B64A9w$ 
Twitter  Bex_16

On 5 Jun 2020, at 09:39, Umar Farook <umarfarook12 at gmail.com<mailto:umarfarook12 at gmail.com>> wrote:

Thanks everyone for the quick response.

Best regards,
Umar

On Fri, 5 Jun 2020, 5:09 am Jay Rai, <jrai at fsu.edu<mailto:jrai at fsu.edu>> wrote:
If your proteins complexes are too small and cannot get the enough contrast with Graphene oxide or in thicker ice then you can give a shot with strepavidine Monolayer.

With kind regards,
Jay
________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of Felipe Merino <felipe.merino at tuebingen.mpg.de<mailto:felipe.merino at tuebingen.mpg.de>>
Sent: Thursday, June 4, 2020 4:12:28 PM
To: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu> <3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>>
Subject: Re: [3dem] [ext] Hexamer dissociate while freezing Cryo-EM grids

Hi Umar,

Or if you know already that your protein is happy on a surface you might want to give graphene or graphene oxide grids a try.

Best,

Felipe

On 04.06.20 21:57, Schacherl, Magdalena wrote:

Hi Umar,

I agree with Jay, that using detergents or surfactants (octyl glucoside 0.1% final conc. or fluorinated octyl maltoside 0.0125% final conc.) might help you. The detergents repel the protein from the water-air interface. Be aware of the fact, that they make the ice thicker and you need a much higher concentration of protein as compared to blotting without detergents.

If blotting per se (the force applied by the contact with filter paper) is the problem, you could try to fix the hexamer with mild glutaraldehyde treatment prior to blotting. We occasionally use 0.5% (v/v) glutaraldehyde (EM grade) and incubate for 10 min at 4 °C, then immediately blot and plunge freeze.

If everything fails, then you might want to try blot-free techniques to apply your protein to grids (e.g. spotting or writing) – there are commercial machines around like Chameleon or VitroJet, or you try to spray your protein (see S. Muench lab or J. Frank lab, and others).

Best regards,

Magdalena

------------------------------------------------------------------------------------------------

Magdalena Schacherl, PhD

Group Leader Structural Enzymology

Charité - Universitätsmedizin Berlin

Institute of Medical Physics and Biophysics  | Charitéplatz 1 | 10117 Berlin

Internal address: Virchowweg 6

Room 03.322, level 3

T: +49 30 450 524196 | F: +49 30 450 524952

magdalena.schacherl at charite.de<mailto:magdalena.schacherl at charite.de>

Website Schacherl lab<https://urldefense.com/v3/__https://biophysik.charite.de/en/research/structural_enzymology_schacherl_lab/__;!!Mih3wA!RCBOFKYqP30RiDg5ED5tufy2vsc_-ByfjO5mvQAv7w-7fIIqLsbhR2y2YIoqQEZRjQ$>

Von: 3dem <3dem-bounces at ncmir.ucsd.edu><mailto:3dem-bounces at ncmir.ucsd.edu> im Auftrag von Umar Farook <umarfarook12 at gmail.com><mailto:umarfarook12 at gmail.com>
Datum: Donnerstag, 4. Juni 2020 um 21:25
An: "3dem at ncmir.ucsd.edu"<mailto:3dem at ncmir.ucsd.edu> <3dem at ncmir.ucsd.edu><mailto:3dem at ncmir.ucsd.edu>
Betreff: [ext] [3dem] Hexamer dissociate while freezing Cryo-EM grids

Dear All,

I would like to ask the community for suggestions on how to overcome the issue of hexamer dissociating while freezing Cryo-EM grids, the negative staining seems to be perfect hexamer at 0.1 mg/ml concentration. But the sample seems to fall apart at 1.5 mg/ml in Cryo freezing, please advise, thank you.

Best Regards,

Umar

_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem<https://urldefense.com/v3/__https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem__;!!PhOWcWs!k48unNWj5DJOh_kn1gA95BSAKgh7Jc1qqGA83iQPUT2OmrJVyx2MRdVMngs8$>

--
Felipe Merino,
Max Planck Institute for Developmental Biology
Department of Protein Evolution
Max-Planck-Ring 5
72076 Tuebingen
Phone: +49 7071 601 1351

ORCID:  https://urldefense.com/v3/__https://orcid.org/0000-0003-4166-8747__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxFxKb4bfg$ <https://urldefense.com/v3/__https://orcid.org/0000-0003-4166-8747__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi4DHBc_cA$>
Publons: https://urldefense.com/v3/__https://publons.com/a/1305699/__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxHZcP5Ycg$ <https://urldefense.com/v3/__https://publons.com/a/1305699/__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi75IBpG2Q$>
Researchgate: https://urldefense.com/v3/__https://www.researchgate.net/profile/Felipe_Merino__;!!Mih3wA!RNVnRe-vD0eh3acekU8RM5IA_xfMhvKD5diRcrMTruE-MteqcC9dzU-ZfxF4NZ_A6Q$ <https://urldefense.com/v3/__https://www.researchgate.net/profile/Felipe_Merino__;!!Mih3wA!Wovm-gVQiGlBAGs2Q9dEbwbScSTOZHyMZnEfw6hf5aactNdJuD44gYtwQi7Xv37Ung$>

_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem
_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20200605/d6829a7d/attachment-0001.html>