Dear Ali,
This is a common problem, and one can indeed tackle it with tilts. There are multiple things going on, which you're probably aware of: (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 basically depend on how well you can deal with them. 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, including in the range that you’re talking about. 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. Good luck!
Regards,
Dmitry
> On Apr 5, 2017, at 9:48 AM, Ali Khan <[log in to unmask]> wrote:
>
> Hi,
>
> Has anyone tried using tilts in a high resolution single particle reconstruction? I am aware of the change in defocus when collecting on a tilted images, but I am wondering if this makes a big difference on a 200-500 kDa protein if the ctf is corrected on each individual particle. This could be a way of dealing with preferential particle orientation.
>
> Best Wishes,
> Ali Khan
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