[3dem] Overcoming orientation preference issue

Thu May 18 20:34:09 PDT 2017

Dear colleagues,

Thank you all very much for so many insightful and detailed suggestions. We
will definitely try to comprehend thess ideas and apply to our project if
possible. It is so nice to have such a wonderful community and thank you
all again for your kind help!

Best,
Yang

On May 18, 2017 2:55 AM, "Marta Carroni" <marta.carroni at scilifelab.se>
wrote:

Dear Yang,
what helped in our case  was to have a thin carbon film (lacey grids +
ultrathin carbon) AND to glow discharge positively in the presence of
pentylamine. Just put a couple of little Whatman paper pieces embedded with
100µl of pentylamine in the glow discharge chamber. You should get a bluish
(rather than pinkish) glow.
Cheers!
Marta

 On 18/05/2017 07:53, Dmitry Lyumkis wrote:

Alas, it is too late for us, since it is post-peer review. But seems like
BioRxiv is really the way to go. Perhaps this is a relevant discussion for
the upcoming GRC or NRAMM workshop.

On May 17, 2017, at 10:23 PM, Gabriel Lander <glander at gmail.com> wrote:

Dmitry, don't make us all wait until August!
http://biorxiv.org/

On May 17, 2017, at 10:51 AM, Dmitry Lyumkis <dlyumkis at salk.edu> wrote:

Dear Yang,

This is a very common problem. Severely preferred orientation not only
limits your ability to reconstruct an accurate map, but can also induce
overfitting, sometimes rather dramatically.

We have a manuscript coming out on this very issue detailing the problems,
both with respect to overfitting, and how one might be able to generally
address it by tilting the stage (as both Pawel and David had mentioned). It
is currently in press, but look out for it in Nature Methods in early
August. Briefly, with severely preferred orientation, I would suggest using
higher tilt angles. Whenever you tilt the stage, there are several things
that you have to take into account: (1) higher background contrast from
inherently thicker ice; (2) increased beam induced movement from tilting;
(3) limitations on estimating the CTF. The first problem is inherent to the
data and cannot be solved (although it can be ameliorated by collecting
using high doses). The second and third problems are essentially practical
in nature and your final resolution will depend on how well you can deal
with them. Definitely use gold grids and not carbon grids (unless there are
better substrates now that further minimize beam-induced motion???). We
have had success using MotionCor2 for movie alignment and GCTF for CTF
estimation (on an “individual” particle basis), but there are certainly
other ways to do things. These combinations have generally been successful
for us with several particle sizes, ranging from 150 kDa to
Megadalton-sized, and provided near-atomic resolution for a range of sizes,
including 150 kDa. For specimens adopting mild preferred orientation, tilts
up to 20-30° are probably good enough; for the pathological cases, 40-50°
might be what you’re looking for. If I had to venture a guess based on what
you’re saying, given the fact that you have 2 preferred orientations, I
would probably suggest somewhere in the 30-40° range (but maybe try 30
first, which will have less beam-induced movement, and will be easier to
reach high-resolution). Some of these details are still being worked out.
Please contact me directly if you would like more information.

Many of the other suggestions that have been proposed may also work. For
example, detergents should help, and many people have had success, but they
require higher protein concentrations to induce the sample to go into
holes. In my personal experience, either with DDM or NP40, one would have
to add detergent to a concentration that is ~CMC (or slightly higher). When
I worked with the HIV trimer, PMC3954647
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3954647/>, I specifically
found that one can induce more random orientations at DDM concentrations of
~2x below the CMC, whereas anything lower than that would not be enough.
Since then, we worked with DDM concentrations at CMC for different samples.
Still, this is going to be specimen dependent, and I have not worked with
Tween20.

Good luck!

Dmitry

Dmitry Lyumkis
Salk Helmsley Fellow
The Salk Institute for Biological Studies
10010 N. Torrey Pines Rd., La Jolla, CA 92037
E: dlyumkis at salk.edu
T: (858)-453-4100 ext. 1155 <(858)%20453-4100>

On May 17, 2017, at 9:24 AM, Jiang,Qiu-Xing <qxjiang at UFL.EDU> wrote:

Dear yang,
Besides other tricks suggested here, in our hands we noticed that our
chemically oxidized carbon films have different surface property from the
glow-discharged films and can change the orientational distribution by
introducing a different interaction profile. A detailed procedure for
chemical oxidation and cleaning is available in https://www.ncbi.nlm.nih.
gov/pubmed/24457027 . It might provide a different alternative.
Best luck.
Qiu-Xing

—
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Yang Li <
yanglixtal at gmail.com>
Date: Wednesday, May 17, 2017 at 8:46 AM
To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
Subject: [3dem] Overcoming orientation preference issue

Dear colleagues,

We have a protein sample that suffers from severe orientation preference,
that most of the particles cluster into two distinct orientations. This way
we have to collect large amounts of data in order to obtain enough
effective particles, which hiders us from reaching high resolution. We have
tried to make thicker ice or adding tiny amount of detergent such as
Tween20, but not working very well so far. I wonder if there are any tricks
we can try to overcome this orientation preference issue? Thank you in
advance for suggestions!

Best,
Yang
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