4 Notes
1. MgCl 2 ∙6H 2 O, CaCl 2 ∙2H 2 O, glycerol, and Tween 80 solutions
are sterilized by autoclaving at 121
C for 20 m. ZnSO 4 ∙7H 2 O,
and sodium pyruvate solutions are sterilized by a filter with
0.22 μm pore size.
2. The 10 nm colloidal gold particle solution should be concentrated by 1.5 times before it is applied onto holey carbon EM
grids.
3. EPU is generally used for data collection of single particle
analysis, but Atlas mode of EPU is useful for scanning the
whole grid.
4. It is preferable to avoid broken hole on the carbon film and
near the edges of the grid for complete data collection to hightilt angle.
5. Select the cell tip area, because the flagellar motor and periplasmic flagella are located at the cell tips in Leptospira spp.
6. Total dose is generally between 100 and 200 e/A ˚ 2 . The number of tilt images is dependent on tilt angles and the angular
increment. For example, tilt series from À60
to 60
with tilt
step of 2
or 3
, there will be 61 and 41 images, respectively.
7. To enhance contrast of the tomogram, use the “Use linear
interpolation” with binning 2 or 4 and “2D Filter” options.
The “Erase Gold” option is better for visualizing periplasmic
flagella. The “Correct CTF” option is better for obtaining
high-resolution subtomogram averaging structure.
8. SIRT reconstructions can generate high-contrast tomogram. It
is better for visualizing periplasmic flagella and particle picking
for subtomogram averaging.
Acknowledgments
The research study described in this chapter was supported by JSPS
KAKENHI Grant Number 18K14639.
References
1. Berg HC (2003) The rotary motor of bacterial
flagella. Annu Rev Biochem 72:19–54
2. Minamino T, Morimoto YV, Kawamoto A et al
(2018) Salmonella flagellum. Salmonella: A
Re-emerging Pathogen. 3–18
3. Izard J, Renken C, Hsieh CE et al (2009)
Cryo-electron tomography elucidates the
molecular architecture of Treponema pallidum,
the
Syphilis
Spirochete.
J
Bacteriol
191:7566–7580
4. Zhao X, Zhang K, Boquoi T et al (2013)
Cryoelectron tomography reveals the sequential assembly of bacterial flagellar in Borrelia
burgdorferi. Proc Natl Acad Sci U S A
110:14390–14395
Visualization of Intact Periplasmic Flagella
137
1. MgCl 2 ∙6H 2 O, CaCl 2 ∙2H 2 O, glycerol, and Tween 80 solutions
are sterilized by autoclaving at 121
C for 20 m. ZnSO 4 ∙7H 2 O,
and sodium pyruvate solutions are sterilized by a filter with
0.22 μm pore size.
2. The 10 nm colloidal gold particle solution should be concentrated by 1.5 times before it is applied onto holey carbon EM
grids.
3. EPU is generally used for data collection of single particle
analysis, but Atlas mode of EPU is useful for scanning the
whole grid.
4. It is preferable to avoid broken hole on the carbon film and
near the edges of the grid for complete data collection to hightilt angle.
5. Select the cell tip area, because the flagellar motor and periplasmic flagella are located at the cell tips in Leptospira spp.
6. Total dose is generally between 100 and 200 e/A ˚ 2 . The number of tilt images is dependent on tilt angles and the angular
increment. For example, tilt series from À60
to 60
with tilt
step of 2
or 3
, there will be 61 and 41 images, respectively.
7. To enhance contrast of the tomogram, use the “Use linear
interpolation” with binning 2 or 4 and “2D Filter” options.
The “Erase Gold” option is better for visualizing periplasmic
flagella. The “Correct CTF” option is better for obtaining
high-resolution subtomogram averaging structure.
8. SIRT reconstructions can generate high-contrast tomogram. It
is better for visualizing periplasmic flagella and particle picking
for subtomogram averaging.
Acknowledgments
The research study described in this chapter was supported by JSPS
KAKENHI Grant Number 18K14639.
References
1. Berg HC (2003) The rotary motor of bacterial
flagella. Annu Rev Biochem 72:19–54
2. Minamino T, Morimoto YV, Kawamoto A et al
(2018) Salmonella flagellum. Salmonella: A
Re-emerging Pathogen. 3–18
3. Izard J, Renken C, Hsieh CE et al (2009)
Cryo-electron tomography elucidates the
molecular architecture of Treponema pallidum,
the
Syphilis
Spirochete.
J
Bacteriol
191:7566–7580
4. Zhao X, Zhang K, Boquoi T et al (2013)
Cryoelectron tomography reveals the sequential assembly of bacterial flagellar in Borrelia
burgdorferi. Proc Natl Acad Sci U S A
110:14390–14395
Visualization of Intact Periplasmic Flagella
137