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<div class="moz-cite-prefix">Dear Yang, <br>
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. <br>
Cheers!<br>
Marta<br>
<br>
On 18/05/2017 07:53, Dmitry Lyumkis wrote:<br>
</div>
<blockquote cite="mid:8D580E70-96F1-4898-A705-FA6D35AA0C00@salk.edu"
type="cite">
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charset=windows-1252">
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.
<div class="">
<div class=""><br class="">
</div>
<div class=""><br class="">
<div>
<blockquote type="cite" class="">
<div class="">On May 17, 2017, at 10:23 PM, Gabriel Lander
<<a moz-do-not-send="true"
href="mailto:glander@gmail.com" class="">glander@gmail.com</a>>
wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
<div style="word-wrap: break-word; -webkit-nbsp-mode:
space; -webkit-line-break: after-white-space;"
class="">
Dmitry, don't make us all wait until August!
<div class=""><a moz-do-not-send="true"
href="http://biorxiv.org/" class="">http://biorxiv.org/</a>
<div class="">
<div class=""><br class="">
<div class="">
<blockquote type="cite" class="">
<div class="">On May 17, 2017, at 10:51 AM,
Dmitry Lyumkis <<a
moz-do-not-send="true"
href="mailto:dlyumkis@salk.edu" class="">dlyumkis@salk.edu</a>>
wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
<div style="word-wrap: break-word;
-webkit-nbsp-mode: space;
-webkit-line-break: after-white-space;"
class="">
Dear Yang,
<div class=""><br class="">
</div>
<div class="">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.</div>
<div class=""><br class="">
</div>
<div class="">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.</div>
<div class=""><br class="">
</div>
<div class="">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, <a
moz-do-not-send="true"
href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3954647/"
ref="aid_type=pmcid" class="">PMC3954647</a>,
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.</div>
<div class=""><br class="">
</div>
<div class="">Good luck!</div>
<div class=""><br class="">
</div>
<div class="">Dmitry</div>
<div class=""><br class="">
</div>
<div class=""><br class="">
</div>
<div class=""><br class="">
<div class="">Dmitry Lyumkis<br
class="">
Salk Helmsley Fellow<br class="">
The Salk Institute for Biological
Studies<br class="">
10010 N. Torrey Pines Rd., La Jolla,
CA 92037<br class="">
E: <a moz-do-not-send="true"
href="mailto:dlyumkis@salk.edu"
class="">dlyumkis@salk.edu</a><br
class="">
T: (858)-453-4100 ext. 1155 </div>
<br class="">
<div class="">
<blockquote type="cite" class="">
<div class="">On May 17, 2017, at
9:24 AM, Jiang,Qiu-Xing <<a
moz-do-not-send="true"
href="mailto:qxjiang@UFL.EDU"
class="">qxjiang@UFL.EDU</a>>
wrote:</div>
<br
class="Apple-interchange-newline">
<div class="">
<div style="word-wrap:
break-word; -webkit-nbsp-mode:
space; -webkit-line-break:
after-white-space; font-size:
14px; font-family: Calibri,
sans-serif;" class="">
<div class="">
<div class="">Dear yang, </div>
<div class="">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 <a
moz-do-not-send="true"
href="https://www.ncbi.nlm.nih.gov/pubmed/24457027"
class="">https://www.ncbi.nlm.nih.gov/pubmed/24457027</a> .
It might provide a
different alternative. </div>
<div class="">Best luck. </div>
<div class="">Qiu-Xing</div>
<div class=""><br class="">
</div>
<div class="">
<div class="">—</div>
</div>
</div>
<span
id="OLK_SRC_BODY_SECTION"
class="">
<div style="font-family:
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text-align: left;
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medium; border-style:
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<span
style="font-weight:bold"
class="">From: </span>3dem
<<a
moz-do-not-send="true"
href="mailto:3dem-bounces@ncmir.ucsd.edu"
class="">3dem-bounces@ncmir.ucsd.edu</a>>
on behalf of Yang Li <<a
moz-do-not-send="true"
href="mailto:yanglixtal@gmail.com"
class="">yanglixtal@gmail.com</a>><br
class="">
<span
style="font-weight:bold"
class="">Date: </span>Wednesday,
May 17, 2017 at 8:46 AM<br
class="">
<span
style="font-weight:bold"
class="">To: </span>"<a
moz-do-not-send="true"
href="mailto:3dem@ncmir.ucsd.edu"
class="">3dem@ncmir.ucsd.edu</a>"
<<a
moz-do-not-send="true"
href="mailto:3dem@ncmir.ucsd.edu"
class="">3dem@ncmir.ucsd.edu</a>><br
class="">
<span
style="font-weight:bold"
class="">Subject: </span>[3dem]
Overcoming orientation
preference issue<br
class="">
</div>
<div class=""><br class="">
</div>
<div class="">
<div class="">
<div dir="ltr" class="">Dear
colleagues,
<div class=""><br
class="">
</div>
<div class="">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!</div>
<div class=""><br
class="">
</div>
<div class="">Best,</div>
<div class="">Yang </div>
</div>
</div>
</div>
</span></div>
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