Resources
Index:
- Cryo-EM Theory & Overview
- Choosing a method based on your sample type
- Sample Preparation
- Grid Preparation
- Sample Screening
- Data Collection
- Data Processing
Cryo-EM Theory & Overview
Grant Jensen Lectures – This YouTube series by Professor Grant Jensen at Caltech provides in-depth lectures on cryo-EM fundamentals from microscope anatomy and Fourier transforms to image formation. It also surveys practical workflows in sample preparation, data collection, and basic data processing in tomography and single particle analysis. There is a companion website where you can access the lecture slides and concept review questions.
Getting Started in Cryo-EM – YouTube
Course Overview – Getting Started in Cryo-EM
CryoEDU – This course provides descriptions and interactive tools for understanding and interpreting single particle and tomography datasets in RELION and CryoSparc. CryoEDU provides access to a cloud desktop running a RELION simulator that users can use to familiarize themselves with the program on a first-come first-served basis.
cryoEDU – Hands-on cryoEM data processing
CryoEM101 – This website provides written explanations, videos, and interactive tools to teach sample preparation, grid preparation, grid screening, data collection, and image processing techniques.
Choosing a method based on your sample type
We will help you determine the best approach for achieving your project goals during your initial consultation. Below is a rough guide for picking a suitable technique for your sample.
Negative Stain is the fastest, lowest-cost way to check if your sample is intact, monodisperse, aggregated, etc. It is ideal for early quality control, troubleshooting, and deciding whether a cryo-EM project is worth pursuing. Negative stain works well for most purified proteins, complexes, viruses, and cells. Very small particles may be hard to see.
Single particle cryo-EM is suitable for obtaining a high-resolution structure of a purified, soluble protein that has a high number of identical particles. Membrane proteins can also be suitable samples for single particle analysis when in detergent. Single particle will provide 2D images of thousands of variably oriented, identical particles that can be back-projected to determine the particle’s 3D structure using programs like RELION or CryoSPARC.
Cryo-electron tomography is ideal when your sample is heterogenous, embedded in a larger environment (such as cells, organells, etc.), or too flexible for single particle. Tomography will provide separate 3D volumes for multiple particles. Non-identical samples can be modeled with segmentation programs like IMOD or Amira. Identical samples can be averaged for structure determination.
FIB-SEM lamella preparation is ideal when your sample is too thick for direct tomography. This may include cells, tissue slices, or thick organelles. FIB-SEM will thin your samples into a lamella suitable for tomography.
Micro-electron diffraction is ideal for protein crystals that are too small for x-ray crystallography and for small molecule structure determination.
Sample Preparation
Protein Sample Preparation for Single Particle Analysis (SPA)
Biochemical preparation and stabilisation - CRYO
ThermoFisher’s Guide to Protein Purification
A Simple Outline of Methods for Protein Isolation and Purification (PDF)
Protein Purification – Chapter 20 from Handbook of Molecular Biotechnology (PDF)
While the revolution will not be crystallized, biochemistry reigns supreme (PDF)
Tomography Sample Preparation
FIB-Milling
Micro-ED Overview
https://myscope.training/CRYO_Introducing_Micro_ED
Grid Preparation
Grid Selection
Handling Grids
Recognizing the carbon side of the grid
Glow Discharge
Preparing hydrophilic grids: Glow discharging Plasma cleaning
Preparing hydrophilic grids: Hydrophobic and hydrophilic grids
Negative Stain
Grant Jensen Lecture (watch from 16:35 to the end)
Negative Staining and Image Classification (download link to PDF)
Negative Stain Demonstration this is a general demonstration. Refer to our facility SOP for detailed guidelines.
Negative staining and image classification (PDF)
Vitrification
Grant Jensen Lecture (watch from 11:34 to 17:28)
Create Biostructure Sample Optimization Guide
Overview of plunge freezing tools
Demonstration of Making Liquid Ethane (watch from 3:08 to 16:26)
Vitrobot Demonstration (watch from 16:26 to 26:05)
Grid Clipping
Example protocol with helpful diagrams We have our own SOP, but the diagrams on this protocol are especially useful.
Demonstration of Loading C-clips into Clipping Tool
Demonstration of Clipping Grids at room temperature
Demonstration of Clipping Grids Under Nitrogen
Sample Screening
Negative Stain Grid Screening
Specimen screening by negative staining - CRYO
Cryo Grid Screening
Specimen screening by cryo - CRYO
Data Collection
Data Collection – Single Particle
Data acquisition in SPA - CRYO
Chapter 4: Cryo-EM Data Collection – Cryo EM 101
Data Collection – Tomography
Data acquisition in Cryo-ET - CRYO
Cryo-ET Chapter 4 – Cryo EM 101
Data Processing
Data Processing – Single Particle
SPA job concept introduction:
MyScope Read pages: motion correction, dose weighting, averaging, particle picking for SPA, 2D classification, 3D reconstruction, and validation.
New users should complete the official RELION and/or cryoSPARC tutorials prior to attempting to process their own data. Working through a standard dataset first builds intuition for how each job functions and makes it easier to apply the basic principles to more complex datasets. In most cases, early preprocessing steps are fairly linear, but later steps are more dataset specific. Due to this variability, it is impossible to provide a fixed roadmap. Instead, users should build foundational understanding with the tutorial datasets.
Data Processing – Tomography
A combination of different programs is necessary for processing tomography datasets. In general, tomography data processing includes five major steps: 1) motion correction, 2) tilt-series alignment, 3) CTF estimation, 4) tomogram reconstruction, and 5) analysis. Our facility uses AreTomo3 for live processing of tomography datasets. This allows us to deliver motion-corrected, CTF-corrected, aligned, and reconstructed tomograms to you shortly after data collection. However, full tomography processing typically requires additional steps such as alignment refinement, denoising, subtomogram averaging, or segmentation. Below is a list of resources to help you get started in understanding your dataset and choosing appropriate programs for downstream processing.
CryoET Chapter 5 – Cryo EM 101
CryoET Chapter 6 – Cryo EM 101
Introducing cryo-electron tomography - CRYO
Cryo-ET Data Processing: Workflow, Advances, and Software Tools - Creative Biostructure
Tomogram Preprocessing Programs:
ETOMO Tutorial (IMOD)
Subtomogram Averaging:
Tomogram Segmentation:
IMOD Segmentation Demonstration
Data Processing Workstations
Processing cryo-EM data requires significant computational resources that standard laptops and desktops don’t typically offer. CryoSPARC’s hardware guidelines is a good place to start if you plan to build your own workstation. If you have questions about specific workstation configurations, please reach out to facility staff or attend our office hours. Contact Us
https://guide.cryosparc.com/setup-configuration-and-management/hardware-and-system-requirements