1.9 Å mouse mFth1 recombinant apoferritin image-shift data collection with image-shift aberration-correction¶

This data collection correct the coma and stigmatism that is linearly dependent on the distance of beam-image shift. It allows quality approaching stage shift approach but even higher image-shift image range than previously acceptable. In this particular setup and microscope, up to 36 1.2/1.3 gold foil holes can be taken in one hl image that requires one auto focus. The pixel size used is significantly smaller than previous setup shown to increase S/N ratio at the same resolution. FSC 0.143 is at 1.92 A.

The resolution and speed gain relative to previous image-shift data collection is from

1. single construct of the recombinant apoferritin and better purification.
2. beam-image-shift aberration correction allows larger shift at the same quality of data collection as stage shift but a fraction of time in stepping through.

IMPORTANT: The number of holes can be imaged depend on the scope-camera configuration. Some scope may show too much image distortion at low magnification for accurate targeting.

Instruments¶

SEMC@NYSBC Krios1

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. 0.66 Å at 37kx scope nominal mag

Grid¶

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

Krios-K2 Presets used for 2.8 A proteasome reconstruction in 5 hours data collection at SEMC @ NYSBC¶

• Nanoprobe was used to allow smaller beam to be used for multiple targeting on 2 um hole although not essential in this case.

Tecnai Krios 300 kV high tension, XFEG, Gun Lens 4 extraction voltage 4100, 70 um C2 aperture and 100 um objective aperture (except when obtaining grid atlas).
Gatan camera dimension 3710(w)x 3838(h) after rotation to Leginon standard. The size of the beam on the scope main viewing screen always covers the 1.2 um hole at en preset on our scope.
Pixel size is 1.10 A at 22500x scope nominal mag.:

Probe	Lens Series	Magnification:	Preset name:	Image Shift (x,y):	Dimension:	Binning:	Camera Dose (e/camera pixel/s):	Exposure Time (ms):	Specimen Dose Rate (e/A^2):	Spot Size:	Defocus (m):	C2 (um):	frame saving:	Camera
micro	LM	100	gr	Aligned	1024x1024	4	n/a	500	n/a	5	0.0	70	no	Ceta
micro	SA	400**	sq	Aligned	927x959	4	8	100	n/a	10	-2e-4	70	no	K2
nano	SA	1300**	hl	Aligned	927x959	4	8	200	0.004	11	-1.5e-4	70	no	K2
nano	SA	22500	fc	0,0	924x924	2	>=8*	500	3.3	8	-7e-7	70	no	K2
nano	SA	22500	fa	0,0	927x959	4	>=8*	250	1.65	8	-1.5e-6	70	no	K2
nano	SA	22500	en	0,0	3710x3838	1	8 e/pixel/s and ~0.7 um on the specimen	7000	46	8	(-1e-6 to -2e-6)	70	200 ms/frame

**These two magnifications are add-on to standard SA lens series made for NYSBC Krios1. May not be available at other scopes.

• Note: If the correlation or power spectrum don't work well enough for the standard Camera Dose Rate of 10 e/pixel/s for fc and fa, you may increase it more so not to have to use longer exposure time. The recommended value in literature is meant to give highest DQE at all sampling frequencies. Using a higher camera dose by 50% will certainly not damage the sensor.

• Note: no defocal pair was acquired in this experiment. Therefore, no ef preset.

Application:¶

MSI-T2 version 3.4 and up (This is similar to older version of MSI-T2 advanced).

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.

Move method to reach the targets:¶

Hole: Presets Manager stage shift
Focus: Presets Manager image shift
Exposure: Presets Manager image shift. Turn on "Correct image shift coma effect" in the Advanced Settings.

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¶

N2 Filling node settings¶

Need to check more often and increase the threshold so that it does not start filling during the 10 minute (25-30 movies) long exposure targeting per hl.

A trick to get Image shift targeting accuracy better¶