Center for high resolution electron microscopy - EM
Preparing specimens for cryo electron microscopy.
About CryoEM in Flemingsberg
The objective of the high resolution electron microscopy core facility is to provide a seamless facility for general EM, cryoEM and high resolution studies of biological specimens. The facility is being located in the Novum building, Flemingsberg.
Examples of specimens being studied are isolated soluble biomolecular complexes, membrane proteins and fabricated systems based on liposomes and nanodiscs. Initial characterization is often made using contrast media (negative staining), while final data is collected from frozen hydrated objects (cryoEM). For membrane proteins 2D crystallization can be made for application of electron crystallography.
The equipment comprises mainly: Three transmission electron microscopes, (one 120 kV with LaB6 filament and two field emission gun microscopes with 200 kV and 300 kV accelerating voltage respectively) all equipped with CCD cameras, a direct electron detector on the 200 kV microscope, preparation equipment for cryo specimens including a plunge freezing robot, dedicated computer hardware and software. Personnel at the center are responsible for the equipment on a daily basis ensuring that ultimate availability and performance of the facility is achieved and for wet lab facilities including equipment for protein purification, 2D crystallization and EM specimen preparation. In addition to support for specimen preparation and data collection, expertise is provided with regard to processing of data.
For projects which are aiming for the SciLifeLab cryoEM national facility the services provided in Flemingsberg can be considered as a valuable entry point to establish conditions for high resolution data collection.
Using the facility
CryoEM in Flemingsberg is generally open to academic and non-academic users. The cost model is fixed for short term screening and data collection projects as follows:
½ day academic institution - 1500 SEK, non-academic – 3000 SEK
1 day academic institution - 3000 SEK, non-academic – 6000 SEK
The fees includes assistance, consumables and microscope time.
The costs for longer continuous projects and for image processing assistance is set depending on the length of the study and the personnel involvement.
Expected feasibility of projects and prioritization is made by the director and managers.
Electron microscopy and image processing:
Wet lab facilities:
Härmark, J, H Hebert, PJB Koeck (2016) Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy. Micron, 85, 39-43.
Nilsson, HE, S Trillo Muyo, M Bäckström, E Thomsson, D Ambort, PJB Koeck, DJ Thornton, H Hebert, GC Hansson (2016) The supramolecular packing of the gel-forming MUC5B and MUC2 mucins and its importance for mucus secretion. J Cystic Fibrosis, 15, S10-S11.
Kim, D., D. Setiaputra, T. Jung, J. H. Chung, A. Leitner, J. Yoon, R. Aebersold, H. Hebert, C. Yip, and J. J. Song (2016) Molecular Architecture of Yeast Chromatin Assembly Factor 1. Scientific Reports, May 25;6:26702. doi: 10.1038/srep26702.
Vijayvargia, R., R. Epand, A. Leitner, T. Y. Jung, B. Shin, R. Jung, A. Lloret, R. S. Atwal, H. Lee, J. M. Lee, R. Aebersold, H. Hebert, J. J. Song and I. S. Seong (2016) Huntingtin’s spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function. eLife, Mar 22;5. pii: e11184. doi: 10.7554/eLife.11184.
*Kumar, R. B., L. Zhu, H. Idborg, O. Rådmark, P-J. Jakobsson, A. Rinaldo-Matthis, H. Hebert and C. Jegerschöld (2016) Structural and functional analysis of calcium ion mediated binding of 5-lipoxygenase to nanodiscs. PlosOne, Mar 24;11(3):e0152116. doi: 10.1371/journal.pone.0152116
Nielsen JT, Kulminskaya NV, Bjerring M, Linnanto JM, Rätsep M, Pedersen MØ, Lambrev PH, Dorogi M, Garab G, Thomsen K, Jegerschöld C, Frigaard NU, Lindahl M, Nielsen NC. (2016) In situ high-resolution structure of the baseplate antenna complex in Chlorobaculum tepidum. Nat Commun. Aug 18;7:12454. doi: 10.1038/ncomms12454.
Mittal M, Kumar RB, Balagunaseelan N, Hamberg M, Jegerschöld C, Rådmark O, Haeggström JZ, Rinaldo-Matthis A. (2016) Kinetic investigation of human 5-lipoxygenase with arachidonic acid. Bioorg Med Chem Lett. Aug 1;26(15):3547-51. doi: 10.1016/j.bmcl.2016.06.025.
Frauenfeld J, Löving R, Armache JP, Sonnen AF, Guettou F, Moberg P, Zhu L, Jegerschöld C, Flayhan A, Briggs JA, Garoff H, Löw C, Cheng Y, Nordlund P. (2016) A saposin-lipoprotein nanoparticle system for membrane proteins. Nat Methods. Apr;13(4):345-51. doi: 10.1038/nmeth.3801.
Koeck PJ. (2016) Annular dark field transmission electron microscopy for protein structure determination. Ultramicroscopy. Feb;161:98-104. doi: 10.1016/j.ultramic.2015.11.010.
Härmark, J., M. K. Larsson, A. Razuvajev, P. J. Koeck, G. Paradossi, L.-Å. Brodin, K. Caidahl, H. Hebert and A. Bjällmark (2015). "Investigation of the elimination process of a multimodal polymer-shelled contrast agent in rats using ultrasound and transmission electron microscopy." Biomedical Spectroscopy and Imaging 4(1): 81-93.
*Kuang, Q., P. Purhonen, C. Jegerschöld, P. J. Koeck and H. Hebert (2015). "Free RCK arrangement in Kch, a putative Escherichia coli potassium channel, as suggested by electron crystallography." Structure 23(1): 199-205.
Kuang, Q., P. Purhonen, T. Pattipaka, Y. H. Ayele, H. Hebert and P. J. Koeck (2015). "A refined single-particle reconstruction procedure to process two-dimensional crystal images from transmission electron microscopy." Microscopy and Microanalysis 21(04): 876-885.
Loureiro A, E. Nogueira, N.G. Azoia, M.P. Sárria, A.S. Abreu, U. Shimanovich, A. Rollett, J. Härmark, H. Hebert, G. Guebitz, G.J. Bernardes, A. Preto, A.C. Gomes, A. Cavaco-Paulo (2015). "Size controlled protein nanoemulsions for active targeting of folate receptor positive cells." Colloids and Surfaces B: Biointerfaces 135: 90-98.
Nogueira E, I.C. Mangialavori, A. Loureiro, N.G. Azoia, M.P. Sárria, P. Nogueira, J. Freitas, J. Härmark, U. Shimanovich, A. Rollett, G. Lacroix, G.J. Bernardes, G. Guebitz, H. Hebert, A. Moreira, A.M. Carmo, J.P. Rossi, A.C. Gomes, A. Preto, A. Cavaco-Paulo (2015). "Peptide anchor for folate-targeted liposomal delivery." Biomacromolecules 16(9): 2904-2910.
Braniste, V., M. Al-Asmakh, C. Kowal, F. Anuar, A. Abbaspour, M. Tóth, A. Korecka, N. Bakocevic, L. G. Ng, P. Kundu, B. Gulyás, C. Halldin, K. Hultenby, H. Nilsson, H. Hebert, B. T. Volpe, B. Diamond, S. Pettersson (2014) The gut microbiota influences blood-brain barrier permeability in mice. Sci. Transl. Med. 6 (263), 263ra158-263ra158.
Kuang, Q., P. Purhonen, C. Jegerschöld and H. Hebert (2014). "The projection structure of Kch, a putative potassium channel in Escherichia coli, by electron crystallography." Biochimica et Biophysica Acta (BBA)-Biomembranes 1838(1): 237-243.
Nilsson, H. E., D. Ambort, M. Bäckström, E. Thomsson, P. J. Koeck, G. C. Hansson and H. Hebert (2014). "Intestinal MUC2 mucin supramolecular topology by packing and release resting on D3 domain assembly." Journal of molecular biology 426(14): 2567-2579.
Poehlmann, M., D. Grishenkov, S. V. Kothapalli, J. Härmark, H. Hebert, A. Philipp, R. Hoeller, M. Seuss, C. Kuttner and S. Margheritelli (2014). "On the interplay of shell structure with low-and high-frequency mechanics of multifunctional magnetic microbubbles." Soft Matter 10(1): 214-226.