About eBIC

The Electron Bio-Imaging Centre (eBIC) was established at Diamond following the award of a £15.6 million grant from the Wellcome Trust, the Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council (BBSRC). The location of eBIC enables scientists to combine their techniques with many of the other cutting-edge approaches that Diamond offers; whilst a partnership with the University of Oxford allows users to access the Polara, a high-containment cryo-electron microscope.

eBIC provides scientists with state-of-the-art experimental equipment and expertise in the field of cryo-electron microscopy, for both single particle analysis and cryo-tomography. Currently eBIC has two Titan Krios microscopes whereas the Polara microscope is located at the University of Oxford. For more information on eBIC please follow this link.

  1. Microscopes
  2. Techniques
  3. Prepare
  4. Beamtime
  5. Post Beamtime
Microscopes -
Titan Krios

Titan Krios I and II are state of the art 300 KeV cryo-electron microcopes from FEI. They are equiped with the latest generation of direct electron detectors, the FEI Falcon II and the Gatan Bio-quantum K2 summit. Both detectors are intergated into FEI's automated data aquisition softwares for single particle and tomography, EPU and TOMO4 respectively.

Polara

The Polara is a 300 KeV cryo-electron microscope from FEI. It is also equipped with a Gatan Bio-quantum K2 summit detector. This detector can be used for semi-automated data collection with SerialEM for both single particle and tomgraphy aplications. The Polara is operable at containment level 3.

Techniques - +
 
Single particle cryo-electron microscopy
This technique requires the collection of a large number of movies from mono-disperse protein complexes or viruses such that their 3D structure can be determined. The Titan Krios and Polara microscopes are equipped with direct electron detectors and automated data collection software that allow a large number of movies to be collected from single particle samples. The type of single particles that can be imaged range from small protein complexes (150 kDa) to large viruses (2 MDa). For proteins smaller than 150 kDa the use of the phase plate may be required and should be discussed with eBIC staff beforehand.
 
Molecular cryo-electron tomography
This technique can be used with single particle samples and is excellent at generating initial models for single particle analysis or for analysing repeating structures in larger pleomorphic structures. Tilt series are collected at areas of interest which can then be aligned and reconstructed to generate 3D reconstructions. For thicker specimens zero-loss imaging is recommended.

Cellular cryo-electron tomography
This technique is used to look at large pleomorphic objects such as vesicles, isolated organelles, bacteria, and intact eukaryotic cells. Tilt series are collected at areas of interest which can then be aligned and reconstructed to generate a 3D reconstructions. For thicker specimens zero-loss imaging is recommended up to a maximum sample thickness is of ~0.5 um.
 
Electron crystallography
This technique requires 2D crystals of lipid-embedded membrane proteins. Untilted and tilted images are collected and then combined to generate a 3D reconstruction of the protein of interest. Electron diffraction can also be done with larger crystals.
Prepare - +
Your grids should be well optimised in advance of your visit to eBIC. We recommend that you bring no more than 16 grids as this is the maximum that will be loaded per 48 hour session. The exception to this are BAG sessions of greater than 48 hour duration. For UK users with a diamond-labelled dry shipper ISPyB can be used to arrange transport to eBIC. For users outside the UK and for non-Diamond labelled dewars you should arrange shipping through your normal university supplier. Further information  on shipping dewars can be found here.
 
If you have the FEI auto-grid loading station we recommend that you clip your grids at your home institution. This will save ~1 hour at the start of your session.
 
Please think about the imaging conditions and detector you want to use prior to your visit as this will save time during your session. If you have any questions please contact us.
 
If you plan to write your data to USB hard drives please bring at least 3 * 2 TB drives.
Beamtime - +

At the microscope

Users of both Krios 1 (m02) or Krios 2 (m03) should meet their local contact at Krios 1 as  grid loading is carried out for both microscopes in lab. 7 of zone 1. The standard user day is listed below:

  • 9 - 9.30: Session starts
  • 9.15 - 10.15: Load up to 8 grids into auto grid cartridges and the Krios cassette
  • 10.20 - 10-30: Load auto grid cassette into the Krios
  • 10.30 – 12: Survey grids to determine the best grid
  • 12 – 12.45: Collect grid atlas image
  • 12.45 – 3: Setup EPU/TOMO4, microscope alignments, detector gain references
  • 3 – 4: Check images, assess CTF and particle density.
  • Automatic data collection for ~40 hours.

EBIC staff will check data acquisition is proceeding from home but the user should also monitor their data collection and email their local contact if they observe issues.

Data transfer

If you have written your data to USB HDDs please make sure that your local contact has the correct mailing address for return postage. Alternatively FTP/Globus may be used to transfer your data from Diamond to your home institution.

Post Beamtime - +

 

Users publishing work containing data from eBIC should ensure they:

  • Acknowledge DLS and any of its personnel in any published material that results from their use of the Cryo-EM facilities. The following statement should always be included in any publications:

“(We acknowledge) Diamond for access and support of the Cryo-EM facilities at the UK national electron bio-imaging centre (eBIC), proposal EM####, funded by the Wellcome Trust, MRC and BBSRC.”
 

  • Notify DLS of the title, authors, facility, journal and reference number, date of acceptance and publication of any scientific publications or papers as a result of their use of the beamtime
  • Notify DLS if they plan to publish any materials e.g. general reports, press releases, etc. that result from their use of the beamtime prior to the material being published and allow time for DLS to review such material and give its prior permission to publish such material if reasonably requested by DLS.
  • The eBIC team outside the building
  • Alex Buzduga and Alistair Siebert with Krios I at Diamond.
  • A cryo EM map of Tobacco Mosaic Virus, with the atomic structure (colours) built into it.
  • Alistair Siebert, Dan Clare, Alex Buzduga, Sonja Welsch and Alan Boswell.
  • Detail of the Adeno virus.
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24/10/2016:

eBIC Director announced: Introducing Prof Peijun Zhang

27/06/2016:

Krios II welcomes its first users from the University of Oxford

29/06/2015:

Krios I welcomes first users from MRC LMB Cambridge

21/01/2015:

[Publication] A national facility for biological cryo-electron microscopy. Saibil, H. R., Grunewald, K. & Stuart, D. I. (2015). Acta Cryst. D 71, 127-135. DOI: 10.1107/S1399004714025280