Services
![hpolk xtal](https://medicine.uams.edu/cmic/wp-content/uploads/sites/39/2024/04/091014-hpolk-xtal-copy-300x212.jpg)
The primary function of the CMIC Structural Biology Core is to facilitate high-quality sample preparation for use in our X-ray crystallography or cryo-EM pipelines.
Services offered by the CMIC Structural Biology Core include:
Sample preparation: protein design, expression, purification, and quality control
A major function of the Structural Biology Core is to help facilitate high-quality sample preparation for projects involving structural biology or biophysical studies, mostly focused on protein production. State-of-the-art equipment for protein expression in bacterial, insect, and mammalian expression hosts is available to core users, including three new stacked Innova 44R culture shakers to be installed for bacterial protein expression, three Multitron shakers with integrated CO2 control, and four CO2 incubators. A tissue culture room with a biosafety cabinet dedicated to structural biology applications is under construction. Additional installations underway include two Beckman JXN26 floor centrifuges, an Avestin Emulsiflex homogenizer, a Branson Ultrasonics SFX250 Sonifier Cell Disruptor, and an Akta Pure 25 L chromatography system in a deli case.
![Pol Iota Structure](https://medicine.uams.edu/cmic/wp-content/uploads/sites/39/2024/04/120404-pol-iota-structure-630x326.png)
X-ray crystallography: crystallization, data collection, and structure solution and refinement
For structure determination by X-ray crystallography, the general pipeline is to first perform crystallization screens with a Mosquito drop-setting robot using standard commercially available kits. Crystallization plates are automatically monitored in a Formulatrix RockImager-1000 plate incubator/hotel. Lead crystallization conditions are optimized by standard methods, such as crystallization grid screens and seeding. Suitable crystals are cryoprotected, flash-frozen, and sent to the SER-CAT facility at APS. UAMS has purchased one share of SER-CAT beamtime and is leasing a half-share from the University of Alabama at Birmingham. Of the 1.5 total SER-CAT shares, 0.5 will be fully available to CMIC members. This half-share provides 12 hours of beamtime on each of the two SER-CAT beamlines during each of three runs per year (72 total hours per year). Data are collected by remote operation of SER-CAT beamlines. X-ray crystal structures are solved and refined using standard software on Linux workstations.
![2D class averages](https://medicine.uams.edu/cmic/wp-content/uploads/sites/39/2024/04/240416-CMIC-2D-class-averages-630x158.png)
Electron microscopy: negative-stain screening, vitrification, data collection, and structure solution and refinement
For structure determination by cryo-EM, the general pipeline is to first analyze samples by negative stain EM using the TF20 electron microscope maintained by the UAMS Digital Microscopy Core. The Structural Biology Core will pay internal instrument charges to access this microscope. Following suitable negative stain screening, an application is initiated to the NIH Transformative High-Resolution Cryoelectron Microscopy program, and samples are frozen to grids with a Vitrobot. Frozen grids can be screened with the TF20 microscope in the UAMS Digital Microscopy Core. The NIH Cryo-EM program has programs with preferred access for IDeA states, and UAMS currently uses an EM grid-clipping system from the Pacific Northwest Center for Cryo-EM within this program. After a sample is allocated time at an NIH center, frozen grids are shipped to the NIH facility, and data are collected by support staff of the NIH center. The resulting data are downloaded to a UAMS storage server in the Structural Biology Core for processing, structure determination, and refinement using a 4-GPU workstation. The Structural Biology Core has budgeted to purchase a 4-GPU workstation dedicated to RPL projects. The Enemark lab also has two 4-GPU workstations, and these computational resources will be available to Core projects when not active on projects of the Enemark lab. Access to cryo-electron tomography techniques will use an analogous pipeline to obtain instrument time and suitable training in the NIH National Network for CryoET. The Structural Biology Core has budgeted funds to purchase microscope time for high-resolution data collection for any cases where instrument time is not obtained in a timely way from the NIH Cryo-EM program.
More Information
For more information or to set up a consultation to discuss research goals, please contact:
Eric Enemark, Ph.D.
Director of the CMIC Structural Biology Core
Email: ejenemark@uams.edu
Rahul Jaiswal, Ph.D.
Manager of the CMIC Structural Biology Core
Email: rjaiswal@uams.edu
How to Acknowledge the CMIC
“Research reported in this [publication, release] was supported (in whole or in part) by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM152281. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”
For abstracts, a short note “Supported by a grant from the NIH/NIGMS – 1P20GM152281” will suffice.