The Cryo-Electron Microscopy Center
The Cryo-Electron Microscopy Center supports industry, academic, government, and non-profit research institutes, providing single-particle and microcrystal electron diffraction capabilities.
Users may choose a combination of these components in order to achieve their scientific goals. A team of personnel will be available to discuss different options and possible deliverables and will offer expert advice on each user’s projects in order to achieve the best results in a timely fashion.
The Cryo-EM Center at HWI provides:
- Expert staff – The Center has experience with multiple users both commercial and academic. The Center staff work with the full research services team at Hauptman-Woodward to ensure the best structural solution is available to our users.
- State of the art instrumentation – A Thermo Fisher Glacios 200 kV microscope with a Volta phase plate, a Falcon 4 Detector for high-throughput grid screening and high-resolution data collection, and a Ceta-D camera for micro-crystal electron diffraction. Thermo Fisher Vitrobot for grid preparation.
- State-of-the-art laboratories – A well-equipped space for biochemical preparation and biophysical characterization of biological samples; a walk-in humidity-controlled environment dedicated for grid preparation.
- Purpose-built facilities – A microscope facility designed to achieve the best performance. Glycol-cooled temperature control, acoustic and electromagnetic isolation, and laminar air flow are implemented into the construction of these spaces. High-capacity data storage and high-performance computational resources with highly tightened data security and user confidentiality are implemented for remote access during and after data collection.
- Timely access -Experimental runs in the Center are scheduled on a weekly basis and can be prearranged ahead of time to suit the special needs of users’ samples when necessary. Users may inspect their grids and data on the fly during data collection and will have remote access to their data for two months after data collection.
The HWI cryo-EM center team can offer advice to help you in sample preparation for successful studies.
For cryo-EM, we broadly follow the same typical protein requirements as for crystallography. Specifically, our recommendations for protein samples to increase the chance of success in cryo-EM are;
- Amount of protein – 50 -100 microliters of protein at 0.5 – 5.0 mg/ml
- Molecular weight of protein – Lower limit: ~50-60 kDa
For the best data we recommend that samples display a single clear band in Coomassie-blue stained SDS-PAGE; and a single band in native blue-gel if feasible, a sharp peak in a suitable SEC column and/or uniformed size in dynamic light scattering or MALS. We can perform standard protein purification before attempting to make cryo-EM grids for analysis.
We can hold a sample preparation meeting with you to discuss the biochemistry of the sample and re-purification methods, etc. If necessary, re-purification and concentration of the sample is done according to protocols established in the sample preparation meeting.
To ensure consistency and reproducibility, your samples will be vitrified on a grid using a Vitrobot (a semi-automatic plunger) in a walk-in humidity-controlled chamber.
Grids will be prepared under different conditions to assess the best conditions for data collection.
Cryo-EM grid screening
Grids will be screened using a Glacios cryo-electron microscope equipped with a Falcon 4 Direct Electron Detector to ensure that single-particle studies or microcrystal electron diffraction can be conducted optimally. Areas on grids will be prioritized for full high-resolution data collection. For single particle analysis, we will determine grid properties including ice thickness, particle density and particle distribution. Grid screening can be combined with overnight data collection.
Automatic data collection and remote data delivery
High-resolution data collection is conducted with the option for users to log into the DMP server at the HWI cryo-EM center via VPN to monitor the data collection process. Raw images and fully pre-processed micrographs are will be saved on HWI’s data storage system for 2 months and made available for the user to transfer the data onto their own servers.
Data analysis and structural determination
The HWI cryo-EM center team can analyze the data and perform structural determination.
Screening for microcrystals for electron diffraction and structure determination
Our Glacios cryo-electron microscope is equipped with the Thermo Scientific MicroED Package and a CETA-D Camera, plus Thermo Scientific EPU-D™ software for screening and automated data acquisition. The HWI cryo-EM center team can analyze the data and perform structural determination.
The who, the how, and the outcome:
Dr. Gaya Yadav
Cryo-EM Center Manager.
Dr. Yadav has almost a decade of experience in using cryo-EM for different scientific projects and has practical experience in applying different techniques and operating different types of cryo-electron microscopes. He is a trained biochemist and structural biologist. His graduate work was on X-ray crystallographic studies of enzymes and his postdoctoral work on the structural and functional studies of macromolecular complexes for both fundamental scientific problems and structure-based drug design. He is the manager of daily operations at the Cryo-EM Center.
Our facilty is purposely designed to produce the highest quality data with our Glacios 200 kV microscope, Volta phase plate, and Falcon 4 and Ceta-D detectors.
We offer a range of services up to and including complete structural solutions.
Publications involving the Cryo-EM Center Group
- Yadav, GP, and Jiang, Q-X. Reconstituted membrane systems for assaying membrane proteins in controlled lipid environments. Chapter 6, pages 93-122. In: “New techniques for studying biomembranes” ed. By Jiang, Q-X. CRC Press, Taylor & Francis Group, LLC. Boca Raton, London & New York (2020).
- Catalysis-dependent inactivation of human telomerase and its reactivation by intracellular telomerase-activating factors (iTAFs). Sayed ME, Cheng, A, Yadav, GP, Ludlow, AT, Shay, JW, Wright, WE, Jiang, Q-X, Journal of Biological Chemistry 294 (30), 11579-11596 1 2019
- Secretory granule protein chromogranin B (CHGB) forms an anion channel in membranes. Yadav, GP, Zheng, H, Yang, Q, Douma, LG, Bloom, LB, and Jiang, Q-X. Life Science Alliance 1 (5), e201800139, 1-18, (2018).
- Current Dilemma on Granin Proteins: Proteins involved in various cellular functions without known mechanisms. Yadav, GP. Cell Cellular Life Science J, 2(2):000115 (2017).
The Cryo-EM Center
Hauptman-Woodward Medical Research Institute
700 Ellicott St., Buffalo,