[3dem] 3dem Digest, Vol 79, Issue 18

Wed Mar 19 12:36:10 PDT 2014

"...Dear colleagues,

I work with HIV  particles and (at least in my samples), they like the 
carbon film on the grids  much better than the holes, but for me this is a big 
problem, as, even using  high titer preps, they rarely go to the holes, 
resulting in less-than-perfect  tomograms. Has anyone else had this problem? What 
did you do to solve it?   

My particles are resusspended in PBS (with gold  fiducials) at the time of 
freezing.

Best,

Luiza..."

Luiza,

The principle difficulty, as mentioned  by some has to do with carbon 
surface charge distribution. As you know, the  longer a carbon film sits, the 
more hydrophobic the surface becomes. Ben's  comment about shorter glow 
discharge times does not make the grid more  hydrophilic but, rather, does not 
alter the hydrophilicity much. Hence, his  suggestion of a very short glow 
discharge time would mean that the surface would  still be quite hydrophobic and 
not wet. The problem resides in the holes  themselves. The edges of the 
holes have a finite thickness depending on the grid  source and, even with 
significant glow discharge, you are not altering the  hydrophobicity of the hole 
edges. We noticed this when looking at lipid vesicles  which were freeze 
quenched. Without fail, the vesicles always went to the hole  edge and were 
unusable for examination.

What  is necessary for success here is that the hole is 'wetable' while  
the carbon surface tends not to be. Both Ben and Qiu Xing have good ideas. 
Here  is another...or at least one more...depends on how long I think about the 
 situation. The longer I think, more options begin to emerge. A key to glow 
 discharging a grid is that only the side you which is up actually  'sees' 
the plasma and is, therefore, charged. As I stated, in our  experience, even 
glow discharging both sides of a holey carbon grid gave us  lipid vesicles 
which liked the hole edges (Qiu Xing will concur since we did  these 
together at one point). Additionally, ice thickness in the hole needs to  be 
non-convex in nature such that the ice is thinner in the center than at the  edges 
of the hole.

So,  here is one way to possibly defeat the issue that you are dealing 
with, in  essence, you are 'pre-wetting' your grid prior to sample application. 
Try not  glow discharging your grids at all or using Ben's suggestion of 
just a very  short time. Just prior to applying your sample, pre-wet your grid  
by applying 3 μl of your PBS buffer containing a very dilute detergent  
concentration...something well below it's CMC (by say 100 fold) and at a  
concentration in which detergent desorption from the  carbon hydrophobic surface 
is very slow because the detergent binds very  tightly to the hole...and the 
carbon surface, of course. Carefully blot the grid  surface and then add 
your virus sample and freeze quench right away. The low  detergent 
concentration effectively makes the hole edges wetable and blotting  wicks/blots away 
the detergent solution on the carbon surface, mimicking Ben's  suggestion.

If  your sample application and freeze quenching occurs quickly after 
blotting away  excess PBS/ Detergent, the detergent molecules forming an 
amphipathic coating on  the hole surface should not have sufficient time to desorb 
from the carbon and  into your HIV envelope coat and, as such, allow you to 
form a nice and even  thickness of ice in the holes.

Just a  thought. I have tried this with success but not with your system 
and/or  conditions.

With my Best  Wishes,

David

David W. Chester,  Ph.D.
Founder and Research Consultant

Analytical BioConsulting, Inc

.
64  Corrigan Avenue 
Meriden, CT. 06268
Phone: (203) 213-4167
Email:  docdave50 at aol.com 

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Today's Topics:

3. Re: Virus  on Carbon (Qiu-Xing Jiang)
4. Re: Virus on Carbon (Ben  Hankamer)

Message: 3
Date: Fri, 14 Mar 2014 11:54:53  +0000
From: Qiu-Xing Jiang <Qiu-Xing.Jiang at UTSouthwestern.edu>
To:  C.J. <biocjh at gmail.com>, 3DEM Mailing List  <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Virus on  Carbon
Message-ID:
<6306D158E60520409F67980750A5AA618AF683 at swmsmail5.swmed.org>
Content-Type:  text/plain; charset="gb2312"

We recently developed chemically modified  carbon films. Protein G on C 
films may help select viral particles through an  antibody. It could allow the 
concentration ~0.1 - 1% of what usually used, and  may be useful. Marc 
Llaguno from my lab is on the Tahoe meeting presenting  this. If you are there, 
please go talk to him. We could send some grids to  you. Best  luck,

__________________________________________________
Qiu-Xing  Jiang
Department of Cell Biology
UT Southwestern Medical Center
Rm  NL5.140B, MC9039
6000 Harry Hines Blvd
Dallas, TX 75390, USA
phone:  214-633-1940  Fax 214-648-5814
Admin Assistant: Ms. Demyia  Pridgen  (phone  214-633-1948)

________________________________________
From:  3dem-bounces at ncmir.ucsd.edu [3dem-bounces at ncmir.ucsd.edu] on behalf 
of C.J.  [biocjh at gmail.com]
Sent: Thursday, March 13, 2014 11:23 PM
To: 3DEM  Mailing List
Subject: Re: [3dem] Virus on Carbon

I also came accross  problems of something like Luiza's. My sample is a  
macromolecule.

Xinghong, whether your method could solve my problem?  Thanks.

2014-03-14 12:16 GMT+08:00 Xinghong Dai  
<bestdz at gmail.com<mailto:bestdz at gmail.com>>:
Hi Luiza,
Maybe  you can try to make the ice a little bit thicker to see if that will 
make any  difference. They theory is that, if the ice is too thin, big 
particles will  not stay in the hole due to size exclusion effect. I had similar 
situation  when working with big viruses.
Good  luck!

Xinghong

Message: 4
Date: Thu, 13 Mar 2014 22:51:11 -0700
From: Ben  Hankamer <b.hankamer at uq.edu.au>
To: Luiza Mendon?a  <luiza at caltech.edu>
Cc: 3DEM Mailing List  <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Virus on  Carbon
Message-ID:  <687303DB-348A-45B1-AA5A-6734FF77E486 at uq.edu.au>
Content-Type:  text/plain; charset=utf-8

Hi Luiza
We have had similar problems with  a number of proteins. What worked for us 
in some cases was to reduce the glow  discharge time. It will depend on 
your set up but try down to 1 sec.  

The rational was to make the carbon sufficiently hydrophilic to allow  the 
ice sheet to form properly in the holes of the holey carbon film, but to  
minimize electrostatic attraction of the proteins to the carbon.  

Ben

> On 13 Mar 2014, at 4:42 pm, "Luiza Mendon?a"  <luiza at caltech.edu> wrote:
> 
> Dear colleagues,
>  
> I work with HIV particles and (at least in my samples), they like the  
carbon film on the grids much better than the holes, but for me this is a big  
problem, as, even using high titer preps, they rarely go to the holes,  
resulting in less-than-perfect tomograms. Has anyone else had this problem?  
What did you do to solve it?
> My particles are resusspended in PBS  (with gold fiducials) at the time 
of freezing.
> 
> Best,
>  Luiza.

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