Aug 25, 2023
Electron microscopy (EM) analysis of LRRK2-Nanotube assembles
- Xinbo Wang1,2,
- Pietro De Camilli1,2
- 11. Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 22. Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Xinbo Wang, Pietro De Camilli 2023. Electron microscopy (EM) analysis of LRRK2-Nanotube assembles. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4bmdzvo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 19, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68889
Keywords: LRRK2, Electron microscopy, Nanotube assemble, ASAPCRN
Abstract
This protocol details methods for the analysis of LRRK2-Nanotube assembles by negative stained EM and Cryo-EM.
Attachments
iuugbvk9p.docx
21KB
Materials
Solutions to prepare:
Low salt buffer:
| A | B | |
| HEPES (7.4) | 20 mM | |
| NaCl | 90 mM | |
| MgCl2 | 2.5 mM | |
| Glycerol | 7% | |
| DTT | 2 mM | |
| GDP | 20 μM |
Negative stained EM analysis
Negative stained EM analysis
Prepare samples in a PCR tube with 300 nanomolar (nM) LRKK2, 20 micromolar (µM) lipid nanotubes and 1 millimolar (mM) GMPPNP.
Incubate samples at 37 °C for 00:30:00 .
30m
Glow-discharge carbon-coated grids (25 mA, 00:00:45 ) during the sample incubation time.
45s
Place the discharged grids on a piece of parafilm.
After incubation, apply 6 µL of the samples to the grid and adsorbed on the grid for 00:05:00 at Room temperature .
5m
Blot the grid with filter paper and stained with 2% uranyl acetate for 00:00:40 .
40s
Dry the grid with filter paper.
Collect images using a Talos L 120C TEM microscope at 80 kV with Velox software and a 4k × 4K Ceta CMOS camera (Thermo Fisher Scientific).
Cryo-EM analysis
Cryo-EM analysis
1h 14m 14s
1h 14m 14s
Dialyze freshly purified LRRK2 into the Low salt buffer.
After dialysis, incubate LRRK2 (2 micromolar (µM) ) with the kinase inhibitor MLi-2 (5 micromolar (µM) ) for 00:10:00 On ice .
10m
Add 20 micromolar (µM) lipid nanotubes into the mixture above and further incubate for 01:00:00 at Room temperature in the presence of 1 millimolar (mM) GTP.
Note
Note: The total volume of the mixture is 12 µL .
1h
Glow-discharge C-flat™ holey carbon gold grids (CF-1.2/1.3-3Au) (15mA, 00:00:45 ) during the sample incubation time, then place the discharged grids on a piece of parafilm.
45s
After incubation, apply 4 µL of the samples to the grid.
Plunge-freeze sample-loaded grids in liquid ethane-propane mixture using a Vitrobot Mark IV (FEI) with the following parameters: blot force, 0; blot time, 00:00:01 ; wait time, 00:00:30 ; drain time, 00:00:00 ; humidity, 100%.
31s
Collect cryo-EM micrographs on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operating at 300 kV, equipped with a post column GIF quantum energy filter and a Gatan K3 Summit DED camera (Gatan, Pleasanton, CA, USA).
Perform the data collection with the SerialEM software. Record movies in super-resolution mode with a physical pixel size of 1.098 A˚ (super-resolution pixel size is 0.549 A˚) and a defocus range of -1 µm to - 3 µm .
Note
The total dose of ~60.6 e− Å−2 was attained by using a dose rate of ~23.5 e− pixel−1 s−1 across 43 frames for 00:02:00 00:00:58 total exposure time. The initial drift and beam-induced motions was corrected using MotionCor2.