[3dem] Overcoming orientation preference issue

Thu May 18 02:55:12 PDT 2017

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 
>> <mailto: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 
>>> <mailto: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 <mailto:dlyumkis at salk.edu>
>>> T: (858)-453-4100 ext. 1155
>>>
>>>> On May 17, 2017, at 9:24 AM, Jiang,Qiu-Xing <qxjiang at UFL.EDU 
>>>> <mailto: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 
>>>> <mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of Yang Li 
>>>> <yanglixtal at gmail.com <mailto:yanglixtal at gmail.com>>
>>>> Date: Wednesday, May 17, 2017 at 8:46 AM
>>>> To: "3dem at ncmir.ucsd.edu <mailto:3dem at ncmir.ucsd.edu>" 
>>>> <3dem at ncmir.ucsd.edu <mailto: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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