Jun 02, 2026
Cryo-EM structural determination of VPS13C
- Dazhi Li1,2,
- Karin Reinisch1,2
- 11Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 22Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Dazhi Li, Karin Reinisch 2026. Cryo-EM structural determination of VPS13C. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqpqpovk5/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: October 19, 2025
Last Modified: June 02, 2026
Protocol Integer ID: 230218
Keywords: Lipid Transport Protein, VPS13, Protein Structure, Cryo-EM, lysosome contact site protein, important for lysosomal membrane damage repair, lysosomal membrane damage repair, em structural determination of vps13c vps13c, organellar membrane, membrane, vps13c vps13c, vps13c, length vps13c, lipid, uncovered novel regulatory mechanism, novel regulatory mechanism
Funders Acknowledgements:
Michael J. Fox Foundation
Grant ID: ASAP-000580
NIH
Grant ID: R35GM131715
Abstract
VPS13C is a ER-lysosome contact site protein that is thought to transport lipids between the two organellar membranes and important for lysosomal membrane damage repair. We employed structure-function analysis of purified VPS13C and uncovered novel regulatory mechanisms. This protocol describes how we obtained the Cryo-EM map for full-length VPS13C and how we build the structural models for VPS13C.
Guidelines
The EM reconstructions for the N-terminal part of VPS13C requires tailored mask creation and local refinement.
Materials
Quantifoil R1.2/1.3 300 mesh gold grids coated with a 2nm continuous carbon film (Cat. # 668-300-AU, Ted Pella).
CryoSPARC v4 or above on HPC or workstation.
Protocol materials
Quantifoil R1.2/1.3 300 mesh gold grids coated with a 2nm continuous carbon filmTed Pella, Inc.Catalog ## 668-300-AU
Safety warnings
VPS13C demonstrates preferred orientation on carbon-coated Cryo-EM grids. But the protein yield could not support freezing on standard gold grids.
Cryo-EM Sample Preparation
9h 15m
Clean the EM grid using an oxygen/argon plasma in a Gatan Solarus Advanced Plasma System.Quantifoil R1.2/1.3 300 mesh gold grids coated with a 2nm continuous carbon filmTed Pella, Inc.Catalog ## 668-300-AU
5m
Apply 3.5 μL concentrated VPS13C sample (~0.04 mg/mL) to the grid and incubate for 30 s.
5m
Blot the grid for 1.5 s at blot force −12 in a Vitrobot Mark IV at 8 °C and 100% humidity, then plunge-freeze in liquid ethane cooled by liquid nitrogen.
5m
Clip and screen the grids, store selected grids in liquid nitrogen.
9h
Cryo-EM Data Acquisition
2d 1h
Load pre-screened grids into a Titan Krios transmission electron microscope operated at 300 kV and equipped with a BioQuantum energy filter and K3 direct electron detector.
1h
Acquire data in SerialEM using super-resolution mode at 0.534 Å/pixel with a defocus range of −2.0 to −2.5 μm.
Note that the defocus was selected for better particle picking. A lower defocus range might be used.
2d
Record each movie for 2.896 s as 50 dose-fractionated frames, corresponding to a total dose of 42.7 e−/Ų.
Collect 4,140 movies at 0° tilt and 4,914 movies at 25° tilt.
This attenuates the preferred orientation problem of the VPS13C sample.
Data Processing using CryoSPARC
1d 17h 20m
Perform motion correction and CTF estimation in CryoSPARC.
Software
CryoSPARC
NAME
Structura Biotechnology Inc.
DEVELOPER
The whole data processing workflow (Strep 9- Strep 18) and the statistics of refined maps.
10h
Manually curate micrographs and discard poor-quality images.
20m
Pick particles and extract them for downstream analysis.
1h
Perform iterative 2D classification to remove junk particles and select good classes.
6h
Generate initial 3D models by ab initio reconstruction.
1h
Perform heterogeneous refinement to separate particle populations and select the best class.
30m
Refine the selected particles by non-uniform refinement.
2h
Apply local or focused refinement with masks to improve flexible regions.
8h
Estimate final resolutions using gold-standard FSC at the 0.143 criterion.
30m