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Research Facilities at the Department of Biochemistry

 

Resources

If you would like a list of publications, scroll to the bottom of the page.

Facility Instruments information

Titan Krios Falcon 3 (TEM mode):

  • magnification 37,000 – 2.26 Angstrom/pixel
  • magnification 47,000 – 1.75 Angstrom/pixel
  • magnification 59,000 – 1.40 Angstrom/pixel
  • magnification 75,000 – 1.09 Angstrom/pixel
  • magnification 96,000 – 0.85 Angstrom/pixel
  • magnification 120,000 – 0.67 Angstrom/pixel

Titan Krios Falcon4i (EFTEM mode):

  • magnification 53,000 – 2.40 Angstrom/pixel
  • magnification 64,000 – 1.96 Angstrom/pixel
  • magnification 81,000 – 1.55 Angstrom/pixel
  • magnification 105,000 – 1.22 Angstrom/pixel
  • magnification 130,000 – 0.96 Angstrom/pixel
  • magnification 165,000 – 0.729* Angstrom/pixel

Talos Arctica Falcon 3 (TEM mode only):

  • 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 size was 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 Falcon 4i detector in counting mode - TBA.
    **AFIS - The Aberration Free Imaging System allows the use of "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

To promote the use and expand our knowledge of the 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: Dima Chirgadze

Next Talk:

TBA

Past Talks:

2023, May 19:
“Relion/cryoSPARC processing” presented by Dr Andrzej Szewczak-Harris, Cryo-EM Facility, University of Cambridge, UK.

2023, April 19:
“Meet The Cryo-Electron Microscopy Facility” presented by Dr Dima Chirgadze, Cryo-EM Facility Manager, University of Cambridge, UK.

2019, November 22:
“In situ liquid phase electron microscopy, 3D imaging and He ion microscopy” presented by Dr Richard Langford, Head of Cavendish Electron Microscopy Suite, University of Cambridge, UK.

2019, November 8:
“The structure of human thyroglobulin” presented by Dr Francesca Coscia, MRC Laboratory of Molecular Biology, Cambridge, UK.

2019, April 10:
“Model building, refinement and validation in CCP-EM” presented by Dr Colin Palmer, CCP-EM Development Team, Rutherford Appleton Laboratory, Didcot, UK.

2019, February 27:
“Micro Electron Diffraction in a Cryo Electron Microscope: A powerful tool for protein and small molecule structure determination.” presented by Dr Abhay Kotecha, Materials and Structural Analysis Division, Thermo Fisher Scientific

2019, January 30:
1) “chameleon: next generation cryoEM sample preparation based on Spotiton” presented by Dr Michele Darrow of TTP Labtech
2) “Cryo-EM Facility update” presented by Dr Dima Chirgadze of Department of Biochemistry, University of Cambridge

2018, April 25:
“Cryo-EM Facility update” presented by Dr Dima Chirgadze of Department of Biochemistry, University of Cambridge

2018, April 18:
“Electron cryo-microscopy of protein transport and assembly machines” presented by Dr Vicki Gold,  Living Systems Institute (LSI) Exeter.

2017, August 10:
“Subtomogram analysis of the 8(!) kDa gas vesicle protein A” presented by Dr Daniel Bollschweiler of Department of Biochemistry, University of Cambridge.

2017, June 6:
“Studying the evolution of macromolecular machines using high-throughput electron cryo-tomography” presented by Dr Morgan Beeby of Imperial College London.

2016, December  6:
“Single-particle CryoEM specimen preparation and data collection — improving on the way” presented by Dr Shaoxia Chen of MRC LMB.

2016, November 3:
“Cryo-electron microscopy with the Volta phase plate” presented by Dr Maryam Khoshouei of Max Planck Institute of Biochemistry.

2016, October 19:
“Optimal single particle cryoEM data collection: microscope, detector, and dose considerations”. Presented by Dr Kasim Sader of FEI

2016, September 13:
“Reducing specimen movement to improve electron cryomicroscopy” presented by Dr Christopher Russo of MRC-LMB.

2016, July 27:
“Sample preparation for Cryo-EM” by Dr Jamie Blaza of MRC-MBU
“”Terms and Conditions” of Cryo-EM” by Dr Dima Chirgadze of Department of Biochemistry.

2016, July 6:
“Titan Krios at The Nanoscience Centre. What to do with your Sundays. Access for University users”  by Dr Dima Chirgadze of Department of Biochemistry.

2016, June 24:
First "Cryo-EM Forum at Biochemistry" Meeting

External links

For those who want to know more about cryo-electron microscopy, we would like to recommend the following resources.

Publications

List of publications which include the data collected on the facility instruments.

  1. Hardwick SW, Stavridi AK, Chirgadze DY, De Oliveira TM, Charbonnier JB, Ropars V, Meek K, Blundell TL, Chaplin AK. Cryo-EM structure of a DNA-PK trimer: higher order oligomerisation in NHEJ. Structure (2023) May 31:S0969-2126(23)00167-3.
    https://doi.org/10.1016/j.str.2023.05.013.
  2. Seif-El-Dahan M, Kefala-Stavridi A, Frit P, Hardwick SW, Chirgadze DY, Maia De Oliviera T, Britton S, Barboule N, Bossaert M, Pandurangan AP, Meek K, Blundell TL, Ropars V, Calsou P, Charbonnier JB, Chaplin AK. PAXX binding to the NHEJ machinery explains functional redundancy with XLF. Science Advances (2023) Jun 2;9(22):eadg2834.
    https://doi.org/10.1126/sciadv.adg2834
  3. Appleby R, Bollschweiler D, Chirgadze DY, Joudeh L, Pellegrini L. A metal ion-dependent mechanism of RAD51 nucleoprotein filament disassembly. iScience. (2023) Apr 25;26(5):106689.
    https://doi.org/10.1016/j.isci.2023.106689
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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
  9. 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
  10. 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
  11. 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.
  12. 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
  13. 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
  14. 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
  15. 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
  16. 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
  17. 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
  18. 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
  19. 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
  20. 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
  21. 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
  22. 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
  23. 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.
  24. 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.
  25. 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
  26. 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
  27. 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
  28. 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.
  29. 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
  30. 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
  31. 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
  32. 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
  33. 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 
  34. 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
  35. 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 

 

 

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