Hauke Hillen
University Medical Center Göttingen / MPI for Multidisciplinary Sciences
General Info
The EMBL Imaging Centre in collaboration with the Heidelberg University is offering a 5-day autumn school (7 – 11 October 2024) covering all aspects of the single particle cryoEM workflow. The autumn school will focus on basics of the workflow aimed at PhD students and early career researchers with no or limited experience in single particle cryoEM.
Content of the course:
- Theoretical background and practicals delivered by experts in the field
- Sample preparation, grid screening and high-end data collection
- Image processing and data analysis
External invited speaker:
Organisers:
Simon Fromm
EMBL Imaging Centre
Simone Mattei
EMBL Imaging Centre
Cristina Paulino
Heidelberg University Biochemistry Center
Contact:
Simon Fromm (simon.fromm@embl.de)
To participate to the autumn school please register by clicking on the following button. Participation is limited to 8 participants. Deadline for registration is August 9th 2024.
Selected participants will be announced by August 16th 2024.
Sponsors:
Lecture/Practical Content
Lectures:
- Dirk Flemming (Heidelberg University): Sample and grid preparation for single particle cryoEM. This lecture will discuss general considerations about suitable samples for single particle cryoEM as well as grid preparation.
- Cristina Paulino (Heidelberg University): The transmission electron microscope. This lecture will cover basics about the instrument itself with a focus on relevant aspects for its use in single particle cryoEM data acquisition.
- Jan Rheinberger (Heidelberg University): Single particle data acquisition. General workflow of single particle data acquisition with a focus on practical considerations for microscope operations.
- Simon Fromm (EMBL): Single particle data analysis. General workflow and concepts of single particle data processing and introduction to the processing software used in the practical.
- Hauke Hillen (University Medical Center Göttingen): Single-particle cryo-EM of organellar and viral gene expression machineries. The lecture will present recent scientific breakthroughs where single-particle cryoEM has been employed.
Practicals:
Practical 1: Sample preparation (Plunge freezing and jet vitrification)
To collect single particle cryoEM data on a cryo-TEM, the biochemically pure sample needs to be embedded into a thin layer of vitrified buffer solution. This is achieved by rapidly plunge or jet freezing the sample after it has been applied onto a support called EM grid.
For this process, different instruments, instrument settings and sample supports (EM grids) can be used to optimize the result. In the practical, different test samples will be vitrified demonstrating the effect of varying those parameters.
The participants will have the opportunity to use:
- Vitrobot Mark 4 (Thermo Fischer Scientific), plunge freezer generating a gradient of ice thickness
- EM GP2 (Leica Microsystems), plunge freezer for single-side blotting
Practical 2: Sample loading and screening with a 200 kV cryo-TEM
After sample vitrification the EM grid is introduced into the cryo-TEM to evaluate the grid preparation. As it is impossible to predict ideal grid preparation settings, it is important to screen differently prepared grids. Modern cryo-TEMs with a so-called autoloader can take up to 12 grids which can then be screened consecutively without need to manually change the sample. For this, grids prepared in the first practical have to be made ready for loading into the autoloader, a process called clipping.
The participants will have the opportunity to use:
- Clipping of EM grids into cartridges
- Loading the clipped grids into 200 kV cryo-TEMs equipped with an autoloader (ThermoScientific Glacios and Talos Arctica microscopes)
- Operation of microscopes to collect screening images using SerialEM software
Practical 3: High-end single particle data collection on a 300 kV cryo-TEM
Once a grid suitable for high-end data collection is identified (practical 2), it is transferred into a different cryo-TEM for high-end data collection. The participants will setup automated data collection to collect a data set suitable for image analysis.
The participants will have the opportunity to use:
- ThermoScientific Titan Krios (300 kV cryo-TEM)
- Monitor on-the fly image processing, which serves to asses data quality and eventually optimize data collection
Practical 4: Single particle data analysis
In this concluding practical, participants will analyze the single particle cryoEM data collected in practical 3 to reconstruct a 3D volume of the molecule of interest, which was used in practical 1 for EM grid preparation.
The participants will have the opportunity to use:
- cryoSPARC software installed on a high-performance computing environment
Programme
Day 1 – Monday, 7th October 2024
| Time (Europe/Berlin) | Agenda and Speaker | Location |
|---|---|---|
| 09:00 – 09:15 | Welcome and Introduction Simon Fromm – EMBL | tbd |
| 09:15 – 10:00 | Round Table Introductions All course participants | tbd |
| 10:00 – 10:15 | Q & A | tbd |
| 10:15 – 10:45 | Coffee Break | |
| 10:45 – 11:30 | Sample and Grid Preparation for Single Particle CryoEM Dirk Flemming – Heidelberg University | tbd |
| 11:30 – 11:45 | Q & A | tbd |
| 11:45 – 13:00 | Lunch Break | EMBL Canteen |
| 13:15 – 18:15 | Practical on Sample Preparation by Plunge Freezing | EMBL Labs |
| 19:00 – | Free evening |
Day 2 – Tuesday, 8th October 2024
| Time (Europe/Berlin) | Agenda and Speaker | Location |
|---|---|---|
| 09:00 – 09:15 | Feedback Discussion and Exchanges on Practicals | tbd |
| 09:15 – 10:00 | The Transmission Electron Microscope Cristina Paulino – Heidelberg University | tbd |
| 10:00 – 10:15 | Q & A | tbd |
| 10:15 – 10:45 | Coffee Break | |
| 10:45 – 11:45 | Practical on Sample Loading on a TEM with an Autoloader Module | EMBL Labs |
| 11:45 – 13:00 | Lunch Break | EMBL Canteen |
| 13:45 – 18:15 | Practical on Grid Screening on a 200 kV TEM | EMBL Labs |
| 19:00 | Free evening |
Day 3 – Wednesday, 9th October 2024
| Time (Europe/Berlin) | Agenda and Speaker | Location |
|---|---|---|
| 09:00 – 09:15 | Feedback Discussion and Exchanges on Practicals | tbd |
| 09:15 – 10:00 | Single Particle Data Acquisition Jan Rheinberger – Heidelberg University | tbd |
| 10:00 – 10:15 | Q & A | tbd |
| 10:15 – 10:45 | Coffee Break | |
| 10:45 – 11:45 | Practical on Single Particle Data Collection and Live Pre-Processing | EMBL Labs |
| 11:45 – 13:00 | Lunch Break | EMBL Canteen |
| 13:15 – 18:15 | Practical on Single Particle Data Collection and Live Pre-Processing (continued) | EMBL Labs |
| 19:00 | Free evening |
Day 4 – Thursday, 10th October 2024
| Time (Europe/Berlin) | Agenda and Speaker | Location |
|---|---|---|
| 09:00 – 09:15 | Feedback Discussion and Exchanges on Practicals | tbd |
| 09:15 – 10:00 | Single Particle Data Analysis Simon Fromm – EMBL | tbd |
| 10:00 – 10:15 | Q & A | tbd |
| 10:15 – 10:45 | Coffee Break | |
| 10:45 – 11:45 | Practical on Single Particle Data Analysis | tbd |
| 11:45 – 13:00 | Lunch Break | EMBL Canteen |
| 13:15 – 18:15 | Practical on Single Particle Data Analysis (continued) | tbd |
| 19:00 | Dinner | Downtown |
Day 5 – Friday, 11th October 2024
| Time (Europe/Berlin) | Agenda and Speaker | Location |
|---|---|---|
| 09:00 – 09:15 | Feedback discussion and exchanges on practicals | tbd |
| 09:15 – 10:00 | Single-particle cryo-EM of organellar and viral gene expression machineries Hauke Hillen – University Medical Center Göttingen / MPI for Multidisciplinary Sciences | tbd |
| 10:00 – 10:15 | Q & A | tbd |
| 10:00 – 10:45 | Coffee Break | |
| 10:45 – 11:45 | Practical on Single Particle Data Analysis (continued) | tbd |
| 11:45 – 13:00 | Lunch Break | EMBL Canteen |
| 13:15 – 13:45 | Feedback and Closing Remarks | tbd |
Practical Info
The course is limited to 8 participants. For selection purposes, please note that your application will not be considered without CV and a letter of motivation, stating your background and how this course would be beneficial for your future work.
Deadline for registration is August 9th 2024.
Selected participants will be notified by August 16th 2024.
There is no registration fee.
Contact:
Simon Fromm (simon.fromm@embl.de)