Facility Instruments information
Titan Krios Falcon 3 (TEM mode):
- magnification 75,000 – 1.06* Angstrom/pixel
- magnification 96,000 – 0.827* Angstrom/pixel
- magnification 120,000 – 0.66 Angstrom/pixel
Titan Krios K3 (EFTEM mode), super-resolution in brackets:
- magnification 26,000 – 3.5 (1.7) Angstrom/pixel
- magnification 33,000 – 2.8 (1.4) Angstrom/pixel
- magnification 42,000 – 2.2 (1.1) Angstrom/pixel
- magnification 53,000 – 1.8 (0.88) Angstrom/pixel
- magnification 64,000 – 1.43 (0.71) Angstrom/pixel
- magnification 81,000 – 1.07* (0.535) Angstrom/pixel
- magnification 105,000 – 0.83* (0.415) Angstrom/pixel
- magnification 130,000 – 0.652* (0.326) Angstrom/pixel
- magnification 165,000 – 0.54 (0.27) Angstrom/pixel
Talos Arctica Falcon 3:
- magnification 17,500 – 6.0 Angstrom/pixel
- magnification 28,000 – 3.7 Angstrom/pixel
- magnification 36,000 – 2.9 Angstrom/pixel
- magnification 45,000 – 2.3 Angstrom/pixel
- magnification 57,000 – 1.82 Angstrom/pixel
- magnification 73,000 – 1.43* Angstrom/pixel
- magnification 92,000 – 1.13* Angstrom/pixel
- magnification 120,000 – 0.89 Angstrom/pixel
- magnification 150,000 – 0.70 Angstrom/pixel
*pixel sizes were confirmed by collecting high-resolution EM data and comparing the obtained reconstruction to the corresponding high-resolution X-ray structure.
Approximate data collection rates (movies/hour):
- Talos Arctica on Falcon 3 detector in linear mode - 50; in counting mode - 22.
- Titan Krios on K3 detector in counting mode with 1 exposure per hole - 70 (non-AFIS) or 300 (AFIS*); 2 exposures per hole - 110 (non-AFIS) or 500 (AFIS).
*AFIS - Aberration Free Imaging System allows using "beam shifts" instead of "stage shifts" during the data collection, thus drastically increasing throughput. The disadvantage is the less accurate positioning of the exposure area within the holes.
K3 gain reference file:
- when correcting gain using the supplied gain reference file, use "Flip Y axis" option in your processing software.
Cryo-EM Forum @ Biochemistry
In order to promote the use and expand our knowledge of Cryo-EM technique, we’ve created a Cryo-EM Forum @ Biochemistry – a series of informal seminars with invited experts in the area of Cryo-EM. Please subscribe to our Cryo-EM mailing list to receive future seminar announcements, Cryo-EM facility updates, and to engage in conversation about the exciting world of Cryo-EM.
Organising team: Andrzej Szewczak-Harris and Dima Chirgadze
Next Talk:
TBA
External links
For those who want to know more about cryo-electron microscopy, we would like to recommend the following resources.
- "Getting Started in Cryo-EM" with Professor Grant Jensen. Video lectures recorded by Grant Jensen:
https://cryo-em-course.caltech.edu (also available on YouTube) - Expansion of "Getting Started in Cryo-EM" series of videos recorded by Grant Jensen and Matthijn Vos:
https://em-learning.com - Lecture series on Electron Microscopy given at the MRC Laboratory of Molecular Biology:
ftp://ftp.mrc-lmb.cam.ac.uk/pub/scheres/EM-course
Publications
List of publications which include the data collected on the facility instruments.
- Islam MS, Hardwick SW, Quell L, Durica-Mitic S, Chirgadze DY, Görke B, Luisi BF. Structure of a bacterial ribonucleoprotein complex central to the control of cell envelope biogenesis.
EMBO J. (2023) Jan 16;42(2):e112574.
https://doi.org/10.15252/embj.2022112574. - Wilson LFL, Dendooven T, Hardwick SW, Echevarría-Poza A, Tryfona T, Krogh KBRM, Chirgadze DY, Luisi BF, Logan DT, Mani K, Dupree P. The structure of EXTL3 helps to explain the different roles of bi-domain exostosins in heparan sulfate synthesis. Nature Communications (2022) Jun 8;13(1):3314.
https://doi.org/10.1038/s41467-022-31048-2. - Chung I, Wright JJ, Bridges HR, Ivanov BS, Biner O, Pereira CS, Arantes GM, Hirst J. Cryo-EM structures define ubiquinone-10 binding to mitochondrial complex I and conformational transitions accompanying Q-site occupancy. Nature Communications (2022) May 19;13(1):2758.
https://doi.org/10.1038/s41467-022-30506-1. - De Bei O, Marchetti M, Ronda L, Gianquinto E, Lazzarato L, Chirgadze DY, Hardwick SW, Cooper LR, Spyrakis F, Luisi BF, Campanini B, Bettati S. Cryo-EM structures of staphylococcal IsdB bound to human hemoglobin reveal the process of heme extraction. Proc Natl Acad Sci U S A. (2022) Apr 5;119(14):e2116708119.
https://doi.org/10.1073/pnas.2116708119. - Sente A, Desai R, Naydenova K, Malinauskas T, Jounaidi Y, Miehling J, Zhou X, Masiulis S, Hardwick SW, Chirgadze DY, Miller KW, Aricescu AR. Differential assembly diversifies GABAA receptor structures and signalling. Nature (2022) Apr;604(7904):190-194.
https://doi.org/10.1038/s41586-022-04517-3 - Kasaragod VB, Mortensen M, Hardwick SW, Wahid AA, Dorovykh V, Chirgadze DY, Smart TG, Miller PS. Mechanisms of inhibition and activation of extrasynaptic αβ GABAAreceptors. Nature (2022) Feb;602(7897):529-533.
https://doi.org/10.1038/s41586-022-04402-z - Liang, S., Thomas, S.E., Chaplin, A.K. Hardwick, S.W., Chirgadze, D.Y. and Blundell, T.L. Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs. Nature 601, 643–648 (2022).
https://doi.org/10.1038/s41586-021-04274-9 - Rebelo-Guiomar P, Pellegrino S, Dent KC, Sas-Chen A, Miller-Fleming L, Garone C, Van Haute L, Rogan JF, Dinan A, Firth AE, Andrews B, Whitworth AJ, Schwartz S, Warren AJ, Minczuk M. A late-stage assembly checkpoint of the human mitochondrial ribosome large subunit. Nature Communications (2022) Feb 17;13(1):929.
https://doi.org/10.1038/s41467-022-28503-5. - Kilkenny, M.L., Veale, C.E., Guppy, A., Hardwick, S.W, Chirgadze, D.Y., Rzechorzek, N.J., Maman, J.D. and Pellegrini, L. Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with DNA polymerase α-primase. Protein Science 31(2):333-344 (2022).
https://doi.org/10.1002/pro.4220 - Dendooven. T., Paris, G., Shkumatov, A.V., Islam, M.S., Burt, A., Kubańska, M.A., Yang, T.Y., Hardwick, S.W. and Luisi, B.F. Multi-scale ensemble properties of the Escherichia coli RNA degradosome. Molecular Microbiology Jan;117(1):102-120 (2022).
https://doi.org/10.1111/mmi.14800 - Yan, Y., Harding, H.P. & Ron, D. Higher-order phosphatase–substrate contacts terminate the integrated stress response. Nature Structural and Molecular Biology 28, 835–846 (2021).
https://doi.org/10.1038/s41594-021-00666-7 - Yin, Z., Burger, N., Kula-Alwar, D., Aksentijević, D., Bridges, H. R., Prag, H. A., Grba, D. N., Viscomi, C., James, A.M., Mottahedin, A., Krieg, T., Murphy, M.P. and Hirst, J. Structural basis for a complex I mutation that blocks pathological ROS production. Nature Communications volume 12, Article number: 707 (2021).
https://doi.org/10.13039/501100009187 - Harris, A., Wagner, M., Du, D., Raschka, S., Nentwig, L.-M., Gohlke, H., Smits, S.H.J., Luisi, B.F. and Schmitt, L. Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5. Nature Communications 12, 5254 (2021).
https://doi.org/10.1038/s41467-021-25574-8 - Munir, A., Wilson, M.T., Hardwick, S.W., Chirgadze, D.Y., Worrall, J.A.R., Blundell, T.L. and Chaplin, A.K. Using cryo-EM to understand antimycobacterial resistance in the catalase-peroxidase (KatG) from Mycobacterium tuberculosis. Structure. Aug 5;29(8):899-912.e4 (2021).
https://doi.org/10.1016/j.str.2020.12.008 - Dendooven, T., Sinha, D., Roeselová, A., Cameron, T.A., De Lay, N.R., Luisi, B.F. and Bandyra, K.J. A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation. Molecular Cell. 81(14):2901-2913.e5 (2021).
https://doi.org/10.1016/j.molcel.2021.05.032 - Spikes, T.E., Montgomery, M.G. and Walker, J.E. Interface mobility between monomers in dimeric bovine ATP synthase participates in the ultrastructure of inner mitochondrial membranes. PNAS 118 (8) e2021012118 (2021).
https://doi.org/10.1073/pnas.2021012118 - Chaplin, A.K., Hardwick, S.W., Liang, S. Kefala Stavridi, A., Hnizda, A., Cooper, L.R., De Oliveira, T.M., Chirgadze, D.Y. and Blundell, T.L. Dimers of DNA-PK create a stage for DNA double-strand break repair. Nature Structural and Molecular Biology 28, 13–19 (2021).
https://doi.org/10.1038/s41594-020-00517-x - Chaplin, A.K., Hardwick, S.W., Kefala Stavridi, A., Buehl, C.J., Goff, N.J., Ropas, Liang, S., De Oliveira, T.M., Chirgadze, D.Y., Meek, K., Charbonnier, J.-B. and Blundell, T.L. Cryo-EM of NHEJ supercomplexes provides insights into DNA repair. Molecular Cell, Volume 81(16):3400-3409 (2021).
https://doi.org/10.1016/j.molcel.2021.07.005 - Grba, D.N. and Hirst J. Mitochondrial complex I structure reveals ordered water molecules for catalysis and proton translocation. Nature Structural & Molecular Biology volume 27, pages 892–900 (2020).
https://doi.org/10.1038/s41594-020-0473-x - Du., D, Neuberger, A., Wu Orr, M., Newman, C.E. Hsu, P.-C., Samsudin, F., Szewczak-Harris, A., Ramos, L.M., Debela, M., Khalid, S., Storz, G. and Luisi, B.F. Interactions of a Bacterial RND Transporter with a Transmembrane Small Protein in a Lipid Environment. Structure, Volume 28, Issue 6, Pages 625-634 (2020).
https://doi.org/10.1016/j.str.2020.03.013. - Oerum S, Dendooven T, Catala M, Gilet L, Dégut C, Trinquier A, Bourguet M, Barraud P, Cianferani S, Luisi BF, Condon C, Tisné C. Structures of B. subtilis Maturation RNases Captured on 50S Ribosome with Pre-rRNAs. Molecular Cell, Volume 80 (2):227-236 (2020).
https://doi.org/10.1016/j.molcel.2020.09.008. - Spikes, T.E., Montgomery, M.G., and Walker, J.E. Structure of the dimeric ATP synthase from bovine mitochondria. PNAS 117 (38) 23519-23526 (2020).
https://doi.org/10.1073/pnas.2013998117 - Nakane, T., Kotecha, A., Sente, A., McMullan, G., Masiulis, S., Brown, P.M.G.E., Grigoras, I.T., Malinauskaite, L., Malinauskas, T., Miehling, J., Yu, L., Karia, D., Pechnikova, E.V., de Jong, E., Keizer, J., Bischoff, M., McCormack, J., Tiemeijer, P., Hardwick, S.W., Chirgadze, D.Y., Murshudov, G., Aricescu, A.R. and Scheres, S,H.W. Single-particle cryo-EM at atomic resolution. Nature 587:152–156 (2020).
https://doi.org/10.1038/s41586-020-2829-0 - Hill, C.H., Napthine, S,, Pekarek, L., Kibe, A., Firth, A.E., Graham, S.C., Caliskan, N. and Brierley, I. Structural studies of Cardiovirus 2A protein reveal the molecular basis for RNA recognition and translational control. BioRxiv 08.11.245035 (2020).
https://doi.org/10.1101/2020.08.11.245035 - Biner, O., Fedor, J., Yin, Z. and Hirst, J. Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration. ACS Synthetic Biology. 9 (6): 1450-1459 (2020).
https://doi.org/10.1021/acssynbio.0c00110. - Rzechorzek, N.J., Hardwick, S.W., Jatikusumo, V.A., Chirgadze, D.Y. and Pellegrini, L. Cryo-EM structures of human CMG–ATPγS–DNA and CMG–AND-1 complexes. Nucleic Acids Research 48 (12):6980-6995 (2020). PMID: 32453425
https://doi.org/10.1093/nar/gkaa429 - Kovtun, O., Dickson, V.K., Kelly, B.T., Owen, D.J. and Briggs, J.A.G. Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles. Science Advances. 6 (30):eaba 8381 (2020). PMID: 32743075
https://doi.org/10.1126/sciadv.aba8381 - Moncrieffe, M.C., Bollschweiler, D., Li, B., Penczek, P.A., Hopkins, L., Bryant, C.E., Klenerman, D. and Gay, N.J. MyD88 death-domain oligomerization determines myddosome architecture: implications for Toll-like receptor signaling. Structure 28 (3):281-289 (2020). PMID: 31995744.
https://doi.org/10.1016/j.str.2020.01.003 - Charenton, C., Wilkinson, M.E. and Nagai, K. Mechanism of 5ʹ splice site transfer for human spliceosome activation. Science 364 (6438):362-367 (2019). PMID 30975767.
https://doi.org/10.1126/science.aax3289 - Wu, Q., Liang, S., Ochi, T., Chirgadze, D.Y., Huiskonen, J.T. and Blundell, T.L. Understanding the structure and role of DNA-PK in NHEJ: How X-ray diffraction and cryo-EM contribute in complementary ways. Progress in Biophysics and Molecular Biology 147:26-32 (2019). PMID: 31014919
https://doi.org/10.1016/j.pbiomolbio.2019.03.007 - Kargas, V., Castro-Hartmann, P., Escudero-Urquijo, N., Dent, K., Hilcenko, C., Sailer, C., Zisser, G., Marques-Carvalho, M.J., Pellegrino, S., Wawiórka, L., Freund, S.M., Wagstaff, J.L., Andreeva, A., Faille, A., Chen, E., Stengel, F., Bergler, H. and Warren A.J. Mechanism of completion of peptidyltransferase centre assembly in eukaryotes. Elife 8 (2019). PMID: 31115337.
https://doi.org/10.7554/eLife.44904 - Yu, Q., Qu, K. and Modis, Y. Cryo-EM Structures of MDA5-dsRNA Filaments at Different Stages of ATP Hydrolysis. Molecular Cell, 72 (6):999-1012 (2018). PMID: 30449722.
https://doi.org/10.1016/j.molcel.2018.10.012