Sub-3Å proteasome image-shift data collection¶

This data collection obtained same quality data set as stage shift at 2.5x speed with the same presets. Even more speed gain relative to super-resolution mode data collection.

The speed gain relative to previous stage shift data collection is from

1. removing iterative movement at Exposure node
2. removing queue processing at Exposure node since both need target adjustment and considerable wait time before stable imaging.
3. 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

Instruments¶

SEMC@NYSBC Krios

1. Scope: FEI Titan-Krios, 300 kV, micro probe at low mag, nano probe at high mags
2. Camera: Gatan K2. mount below Falcon/Ceta. 1.21 Å at 29kx scope nominal mag

Grid¶

• 1.2/1.3 C-flat carbon support; 400 mesh Copper grid.

Krios1-K2-NYSBC

Application:¶

MSI-T2 Advanced (= MSI-T + "Beam Tilt Image" for coma-free alignment + "N2 Filling" for Krios auto nitrogen filler)

Presets Manager:¶

No preset cycling (Krios has its own normalization).

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

Move method to reach the targets:¶

Hole: Navigator Iterative movement: Target Tolerance: 1e-7 m; Acceptable Tolerance: 3e-7 m
Focus: Presets Manager image shift
Exposure: Presets Manager image shift

Wait time before final exposure:
3 seconds
10 seconds addition for the first target¶