Sub-3Å proteasome image-shift data collection » History » Revision 5
Revision 4 (Anchi Cheng, 08/16/2016 07:39 PM) → Revision 5/8 (Anchi Cheng, 08/16/2016 07:45 PM)
h1. Sub-3Å proteasome image-shift data collection
This data collection obtained same quality data set as stage shift at 1.4x speed with the same presets. More speed gain relative to super-resolution mode data collection. Final reconstruction from the better of the two 3D subclasses (48,000 or ~57,000 cleaned-up particles) gave FSC 0.143 at 2.89 A.
The speed gain relative to previous stage shift data collection is from
# removing iterative movement at Exposure node
# removing queue processing at Exposure node since both need target adjustment and long wait time before stable imaging.
# using counting mode. not super-resolution mode on K2.
*VERY IMPORTANT*: Measure the amount of beam tilt associated with the image-shift at your scope so that it is not compromising the data. Our scope is at 0.3 mrad at the 1.7 um image shift we applied in this experiment.
The Transfer Function will reduce all structure factor to 78% at these beamtilts assuming 0.167 mrad per 1 um image shift:
|image shift (um) |45 degree phase modification resolution(0.78 transfer function amplitude) (Angstroms)|
|1|2.4|
|2|3.0|
|3|3.5|
|4|3.8|
h2. Instruments
SEMC@NYSBC Krios
# Scope: FEI Titan-Krios, 300 kV, micro probe at low mag, nano probe at high mags
# Camera: Gatan K2. mount below Falcon/Ceta. 1.21 Å at 29kx scope nominal mag
h2. Grid
* 1.2/1.3 C-flat carbon support; 400 mesh Copper grid.
{{include(Krios1-K2-NYSBC)}}
h2. Application:
MSI-T2 Advanced (= MSI-T + "Beam Tilt Image" for coma-free alignment + "N2 Filling" for Krios auto nitrogen filler)
h2. Presets Manager:
No preset cycling (Krios has its own normalization).
h2. Queuing usage:
Hole Targeting: Queuing, target at the carbon area to cover four holes.
Exposure Targeting: *No Queuing*. automated targeting. See [[Exposure Targeting Set-up for MSi-T for Four Holes]]
h2. Move method to reach the targets:
Hole: [[Iterative_Stage_Movement|Navigator Iterative movement]]: Target Tolerance: 1e-7 m; Acceptable Tolerance: 3e-7 m
Focus: Presets Manager image shift
Exposure: Presets Manager image shift
h2. Wait time before final exposure chosen to reduce maximal frame drift to average to 2 Angstroms :
3 seconds
10 seconds addition for the first target
h2. A trick to get Image shift targeting accuracy better:
{{include(Scaling [[Scaling Image Shift Matrix Calibration)}} Calibration]]
Get a better estimate of stage position matrix at en magnification by scaling the matrix at hl mag.
You can find the matrix in image report (Click on the "i" in the myamiweb imageviewer toolbar)
Scale the matrix by magnification will give a reasonable value.
For example, if your hl preset at 1300x has a matrix of
<pre>
2e-9 -6e-9
-6e-9 -2e-9
</pre>
Then a good estimate of the matrix for your en preset at 22500x would be multiplied by 1300 / 22500 and becomes
<pre>
1.16e-10 -3.47e-10
-3.47e-10 -1.16e-10
</pre>
Enter this in Matrix node in Calibrations application using the Edit tool in the toolbar