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Single particle cryoEM

LU has started to build an infrastructure for single particle cryoEM

We are happy to announce that Lund university has granted funding for the first phase of building a single particle cryoEM infrastructure! The present funding is mainly aimed to provide expertise in the form of a single particle cryoEM application expert and reducing costs of using the existing screening microscope at nCHREM. 

 

Flavi virus

What is single-particle cryo-em?

Single-particle cryo-electron microscopy (cryoEM) is a rapidly growing field in modern structural biology that allows the atomic resolution structure determination of large macromolecular complexes without the need to crystallize them, a prerequisite and limitation for X-ray crystallography.

Get started

A rapidly growing community of researchers at Lund University is applying cryoEM to a wide range of biological questions. High-resolution structural information can only be obtained with very high-performance microscopes and detectors that LU does not at present have. CryoEM also requires extensive sample optimization and prescreening of samples before final data collection. LU researchers are thus highly dependent on SciLifeLab facilities in Stockholm and Umeå for both pre-screening and data collection. To get in touch with the Stockholm cryo-EM facility staff (Marta Carroni, Julian Conrad, Dustin Morado, Karin Walllden, José Miguel de la Rosa and Stefan Fleischmann) use thiscryoEM_stockholm_lab [at] scilifelab [dot] se ( SciLife Lab cyroEM email.)

Sample optimatization and prescreening at nCHREM in Lund

Funding is now in place to facilitate screening of cryoEM samples at LU’s only suitable electron microscope, the cryoTEM at the National Centre for High Resolution Electron Microscopy (nCHREM) at Kemicentrum. This is a prerequisite for future data collection on the state-of-the-art microscopes in Stockholm, Umeå or elsewhere, and an essential step to secure and strengthen the user community at LU within a method that is revolutionizing modern structural biology

The Cryo-TEM facility at nCHREM has been in operation as an open facility since 1996, and thus has one of the longest track records in Sweden of cryo-plunging, using the method developed by Jacques Dubochet (Nobel Laureate 2017). With the installation of the new CryoTEM instrument in 2016 (JEM-2200FS), funded by LTH and Science Faculty infrastructure grants, we have good facilities for a screening process. Due to a rapidly increased demand from other users in the field of colloidal chemistry and electron tomography, access to the microscope at nCHREM is currently being limited to one day per week, which will be set aside for solving the imminent problem for existing biology users and to kick-start the growth of the community at LU. However, sample preparation equipment will be available on other days. The AE will be available one day a week for microscope operation, a further 1-2 days for sample preparation and the remaining time will be devoted to microscope maintenance and image processing. The AE will be available free of charge for the users for microscopy and specimen preparation.

Pål Stenmark (Faculy of Medicine), Derek Logan (Faculty of Science) and Reine Wallenberg (Faculty of Engineering/LTH) were the main applicants and the application work was coordinated by MicLU

The existing single particle cryoEM Lund university BAG has been coordinated by Lotta Happonen and Derek Logan. With the recruitment of the singple particle cryoEM expert Markel Martinez Carranza the LU BAG coordination is now further strengthened. Since Markel is leaving LU for another assignment we have engaged emil [dot] tykesson [at] med [dot] lu [dot] se (Emil Tykesson) and crispin [dot] hetherington [at] chem [dot] lu [dot] se (Crispin Hetherington) to assist the cryoEM in Life Science user community.

People that are interested in screening samples at LU are welcome to contact primarily crispin [dot] hetherington [at] chem [dot] lu [dot] se (Crispin Hetherington), microscope technician and Reine Wallenberg, director at nCHREM to allocate time for single particle cryo-EM usage. We have also set up two workstations at BMC for the users to process their data, as well as a storage dewar for saving grids and a dry shipper. For those that want to use the workstations or get help with analysis, we recommend that you contact emil [dot] tykesson [at] med [dot] lu [dot] se (Emil Tykesson). Even if Emil will have a focus on the data analysis part while Crispin will assist in the parts done at nCHREM there will to some extent also be an overlap in what they can help you with. It is therefore recommended that you send your email with request for help adressed to both Emil and Crispin if you are not sure who of them that is most suited to help you.

For access to resources at LUNARC contact CIPA application expert anders [dot] sjostrom [at] lunarc [dot] lu [dot] se (Anders Sjöström) that is our primary contact at LUNARC.

How to get started in Lund - Cryo-EM single particle analysis

Cryoelectron microscopy has recently seen a revolutionary increase in the resolution it can yield when looking at single macromolecules. It is best suited for particles above 100 kDa that are difficult to crystallize, and generally requires less material than X-ray crystallography.

In order to obtain a high-resolution structure, the main challenge to overcome is the sample preparation process. Particles are suspended in a meniscus of non-vitreous ice that is formed in the holes of a carbon film within a copper grid. A grid where the particles are evenly distributed in the holes is needed for best results, with thin ice for best contrast. The grid blotting process can be challenging, and it needs to be optimized until a suitable grid is found. Each grid requires 3 µl of sample, at a concentration somewhere between 0.1 mg/ml - 5 mg/ml depending on the nature of the sample.

Before microscope time is allocated, the sample should be purified in as low salt and glycerol concentrations as possible, in order to maximize the contrast. It is very useful to image the sample via negative stain to have an idea of the quality of the sample, and whether it is aggregated or not. Well distributed particles in a negative stain grid indicate that the sample is likely to succeed in later stages of the process. It is also possible to screen Cryo-EM grids with the microscope at nCHREM.

Once Cryo-EM microscope time has been allocated at SciLifeLab, the sample will be screened in a Talos Arctica microscope, and if a suitable grid is found a dataset will be collected. Suitable grids will also be stored for a later data collection in the more powerful Krios microscope.

If you are interested in Cryo-EM SPA, you can contact Emil Tykesson or Crispin Hetherington who works partly as a Cryo-EM application experts at Lund University.

Hardware

Two workstations are currently available for processing cryo-EM data, for the use of any interested Lund University users. These machines are equipped with two high end GPUs each, allowing for fast parallel processing using software like Cryosparc or Relion. In order to get access to these computers, you can contact Emil Tykesson.