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<div class="moz-cite-prefix">Dear Yang,<br>
glow discharging Quantifoil holey carbon grids with an additional
thin carbon film in presence of pentylamine just helped also with
our protein in the past.<br>
As Marta described ... very helpful!<br>
For the side views normal glow discharging without pentylamine,
for the top views glow discharge in pentylamine environment.<br>
Good Luck!<br>
Jörg<br>
<br>
<br>
<br>
<br>
<br>
On 18.05.2017 11:55, Marta Carroni wrote:<br>
</div>
<blockquote type="cite"
cite="mid:17dd4c13-c2d6-c879-e1d8-b2d28c1e71ed@scilifelab.se">
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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">
<meta http-equiv="Content-Type" content="text/html;
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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class=""> <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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<pre class="moz-signature" cols="72">--
****************************
Jörg Bürger
Charité - Universitätsmedizin Berlin
Institut für Medizinische Physik und Biophysik
Charitéplatz 1, CCO - Virchowweg 6
10117 Berlin
Max Planck Institut für molekulare Genetik
Ihnestr. 63-73
14195 Berlin
tel. 030-8413-1644/1278
fax. 030-8413-1385
email. <a class="moz-txt-link-abbreviated" href="mailto:buerger@molgen.mpg.de">buerger@molgen.mpg.de</a></pre>
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