Equipment & Methodology

In our space located on the fifth and second floors of the Black building, we have all the equipment to prepare our samples, collect the EM data and analyze them.

Grid Preparation:

  • Edwards Auto 306  —  Turbo-pumped carbon evaporation system. This tool creates a clean, hydrophilic and non-reactive surface which is needed for making frozen-hydrated grids of labile macro-molecular complexes.
  • Gatan Solarus —  This plasma cleaning device creates a clean hydrophilic surface for our frozen- hydrated samples.
  • Pelco easiGlow 9100 — This plasma cleaning device creates a clean hydrophilic or hydrophobic surface for our frozen hydrated samples.
  • Manual cryo plunging unit — With this hand-built apparatus, frozen-hydrated samples are produced by manual two-sided blotting, and release of a gravity-operated plunger.  Since humidity and temperature are not controlled, reproducibility is not strictly ensured.
  • FEI Vitrobot Mark IV Vitrification Robot — This cryo-plunging system, allows control of parameters such as blotting time, relative humidity and temperature for reproducible results even by inexperienced users.
  • Gatan CP3-GB Vitrification system — Users of this cryo-plunging system can choose one- or two- sided blotting and can set the blotting time.  The humidity can be monitored and the cryogen temperature regulated with this system.

 

Electron Microscopes:

  • FEI Tecnai F20 — Cryo TEM with Gatan 4x4k Ultrascan CCD camera and CT3500 and Gatan 914 High tilt cryo-transfer systems. This microscope operates with voltages up to 200 kV and has a highly coherent electron FEG source for high-resolution data collection from frozen-hydrated samples. A number of software packages have been installed to allow automated data collection.
  • FEI Tecnai Polara F30 — Cryo TEM  with Gatan K2 Summit direct electron counting camera. This microscope operates with voltages up to 300 kV and is capable of operating at liquid helium or liquid nitrogen temperatures. As the F20 it is equipped with a highly coherent FEG source. The instrument has a very stable cartridge style system that allows up to 6 samples to be loaded at once for screening. Once the best sample has been found, data can be collected for multiple days with little contamination or operator intervention using the program Leginon.  Vibration and field canceling systems are also installed.

 

Software for Data Collection on the Microscopes:

 

Data Digitization and Screening:

With the installation of the Gatan K2 Summit our lab no longer collects on film but we still have the systems below available.

  • SIRA Digital diffractometer.  Micrographs on film can be quickly screened for image quality using this apparatus.
  • ZI Photoscan 2001. This high-resolution (7 µm) scanner allows micrographs collect on film to be digitized with great accuracy for high-resolution results.
  • Three Nikon Super Coolscan 9000ED Film Scanners

 

Data Processing:

A.  Software

B.  Computing Facilities

  • Micrograph Storage Server — Micrographs generated from Gatan K2 direct director are sent to Catalina server via a 10Gb high through put direct Ethernet connection. Catalina can store up to 33TB of data in raid 6 configuration which can tolerate two concurrent hard drive failures.
  • Preprocessing Server – Midway server comprises of 32 cores (Intel Xeon(R) CPU E5-4620), 256GB ram, 24TB raid 6 storage. This server handles tasks such as CPU frame alignment, automated particle picking, defocus calculation, etc.
  • Preprocessing GPU Servers – GPU01 and GPU02 servers, equipped with Nvidia GTX 680 and Nvidia GTX770 GPU cards respectively, provide another option for micrograph frame alignment at approximately 30sec per 600MB stack.
  • Computation Cluster 1 – Installed in 2008, comprises of 120 computing nodes, each with 8 processing cores and 32GB ram, based on AMD Opteron 2352 at 2.1GHz. Heavy computational tasks such as classification, reconstruction and refinements are submitted here.
  • Computation Cluster 2 – Acquired in 2013, comprises of 24 computing nodes, each with 4 processing cores and 32GB ram, based on Intel Xeon CPU E3-1240 V2 at 3.40GHz. Relion computation jobs are better suited on this type of cluster.
  • Simulation and Modeling Cluster – This is a high density Twin-blade cluster comprises of 16 nodes, each with 32 processing cores and 64 GB ram based on AMD Opteron(TM) Processor 6274 at 2.2GHz. Molecular Dynamics Flexible Fitting (MDFF) and Assisted Model Building with Energy Refinement (AMBER) jobs are submitted here.
  • Data Storage Servers – Four main file servers for general and long term data storage for a total of  >  120TB.
  • 3D Visualization workstation – Three 3D workstations based on the Nvidia 3D Vision technology.
  • 3D printer – uPrint 3D printer of Stratasys produces physical 3D objects using thermoplastic material.