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About the Facility

At present we have four transmission electron microscopes (TEMs), one multimode scanning probe microscope and one scanning electron microscope (SEM) at the nCHREM. Three of the TEMs have cryo-stage for liquid nitrogen and one have also helium cooling possibilities.

Cryo Electron Microscopy

The aim for the cryo electron microscopy facility is to provide state of the art equipment and expertise to enable external research groups to take on projects in biological cryo electron microscopy. Dependent on the needs and expertise level of our customers, the equipment can be made available either on an 'as is' basis or with various degrees of involvement in microscopy and image processing from our staff.

Cryo electron microscopy (cryo EM) is an important tool for high resolution structural studies of biological macromolecules that don't readily form three dimensional crystals. This is often the case for membrane proteins and large molecular assemblies. It is also the case for short lived intermediate states which are often possible to trap by the rapid freezing technique used for cryo EM sample preparation.

In the case of membrane proteins, two dimensional (2D) crystals, suitable for studies by cryo EM are often easier to obtain than three dimensional crystals which are required for X-ray crystallography. The small amounts of protein which are needed is an advantage compared to both X-ray crystallography and NMR techniques. In some cases, the 2D crystals are large and well ordered enough to allow the deduction of the atomic structure of the studied protein, whereas in other cases, the final result is a 3D reconstruction where only the secondary structure elements are resolved.

A distinct advantage of cryo EM is the possibility to image individual molecules (in projection). This 'single particle' technique is often the only option for structural studies of large macromolecular assemblies when size and difficulties in obtaining 3D crystals make other techniques impractical. In fortunate cases, it has been possible to obtain a resolution comparable to what is attainable by cryo EM 2D crystallography whereas in other cases difficulties in imaging a sufficient number of particles or other technical limitations yields a resolution where only the molecular envelope can be determined.
Even in those cases, cryo EM is a valuable source of information when one or more of the components of a macromolecular assembley are of known structure. These components can then be docked in the molecular envelope and the quaternary structure of the complex can be deduced.
Special techniques exist to analyse cases when the molecules under study form helices or have a high point group symmetry.

The Swegene cryo electron microscopy center which is part of nCHREM (National Centre for High Resolution Microscopy) in Lund is currently (March 2003) having a JEOL 3000SFF, including a Helium cooled cryo stage and a field emission gun installed on our premises. The microscope is equipped with a 4096 by 4096 pixels Tietz CCD camera for rapid data collection. The combination of a highly coherent electron source and specimen preservation at liquid helium temperature give opportunities for data acquisition at very high resolution.

Within nCHREM, there is equipment for specimen preparation as well as a Philips CM120 microscope suitable for specimen screening. We also have access to a Zeiss SCAI high resolution scanner.

Enquiries can be made to:

Professor Hans Hebert
Phone: +46-(0)46-222 81 26
E-mail: hans.hebert@mbfys.lu.se

 


Electron diffraction pattern of a microsomal glutathione transferase 2D crystal at 45° tilt.

 


The 6Å map of microsomal glutathione transferase 1 as seen from the lumenal side of the ER membrane.

 

A 3D reconstruction of peroxisomal alcohol dehydrogenase from Pichia pastoris. The assembly possesses tetrahedral symmetry.
The reconstruction was performed by single particle techniques from images in vitreous ice. A homologous molecule with known crystal structure (1GAL)
is modeled into two of the lobes.