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Fesik Lab

Facilities

Laboratory. The Fesik lab consists of about 4,000 sq. ft. with bench space and seating capacity for 28 researchers. The lab contains all the necessary equipment to perform molecular and cell biology experiments, protein purifications, sample preparation, and medicinal chemistry. It contains eight fume hoods, a cell culture room with four biohoods, a room housing the fermentor, and a shared cold room. In addition, the Fesik Lab has access to NMR spectrometers, X-ray equipment, and a crystallization robot in the structural biology core facility, instrumentation in the High Throughput Screening Core and Mass Spectrometry Core, and additional equipment in the Vanderbilt Institute of Chemical Biology’s Chemical Synthesis Core and other chemistry labs on campus (Professors Sulikowski, Lindsley, and Johnston).

Shared facilities. The Fesik Lab has access to the services and expertise available in the many Core facilities that Vanderbilt maintains to supplement the available capabilities of the Fesik Lab and others. Among these are the Mass Spectrometry Core, the High-Throughput Screening Core, and the Chemical Synthesis Core. The High-Throughput Screening facility, directed by Dr. David Weaver, is designed to support industrial-scale laboratory automation and is fully-equipped with state-of-the art readers, liquid handling, laboratory automation, and laboratory information management systems (LIMS) needed to perform the complex cell-based and cell-free assays to support Vanderbilt investigators in basic and translational research. The HTS Core also maintains the Vanderbilt screening collection, which currently numbers more than 160,000 discreet compounds. The Chemical Synthesis Facility, directed by Dr. Gary Sulikowski and staffed with full-time synthetic and medicinal chemists, supports the Vanderbilt community in all aspects of medicinal chemistry and organic synthesis. This fee-for-service core provides quantities of known or unknown compounds with an emphasis on parallel synthesis of small libraries, HPLC purification, and scale-up of synthetic procedures.

Computer. To process and analyze structural data we have three Dell T3500, 8 processor Xeon 3.2 GHZ linux workstations equipped with 16 GB of memory, Nividia graphics boards and stereoscopic glasses. Each scientist has an Apple workstation (e.g. IMac, 3.06 GHz Intel Core 2 Duo processor with 4GB memory) on his or her desktops. The NMR and X-ray software is maintained and updated through membership in the Structural Biology Grid at Harvard Medical School (SBGrid) which is a computing collaboration of more than 120 X-ray crystallography, NMR, and electron microscopy laboratories. This software includes all of the major NMR packages (e.g. NMRPipe, Xplornih) and Xray packages (e.g. CCP4, HKL-2000, Phaser, Buster TNT). The modeling software such as Dock (UC San Francisco), Sybyl (Tripos), InsightII (Accelrys), MOE (Chemical Computing Group), and open source tools like Autodoc and Pymol are accessed through site licenses of the Vanderbilt Structural Biology Computing group. Pipeline Pilot is obtained through a shared license with the Vanderbilt Institute of Chemical Biology (VICB) HTS group.

EQUIPMENT

Protein preparation. The lab contains all the necessary equipment to clone, express, and purify proteins such as an Eppendorf MasterCycler for running PCR and two E-Gel iBase and E-Gel Safe Imager systems for DNA agarose gel electrophoresis and collection. Other related equipment includes: a -80 °C freezer to store competent cells and proteins; a -20 °C freezer to store enzymes, plasmids and reagents; a bench-top and floor model refrigerated shaker/incubator; a BioFlo 415 fermentor for cell culture growth; bench-top and floor model centrifuges for cell harvesting; and a APV 2000 cell homogenizer for lysing cells.

Large scale protein purifications are carried out using two AKTA Purifiers and an FPLC set up in chromatography refrigerators along with a collection of high volume columns including Ni-NTA, Glutathione Sepharose, SP Sepharose, Heparin Sepharose, Q Sepharose, Sephacryl S100 and S200, Superdex 75, Jupiter C4, G25 desalting and HIC. Protein quantization is carried out with the NanoDrop 2000c using A208, Pierce BCA and Pierce 660. Proteins are concentrated using Amicon cells ranging from 4 mL to 380 mL in capacity.  

X-ray crystallography. Crystallization screens are designed and prepared using the Hamilton Microlab Star liquid handling workstations, and protein crystallization drops are set up with the nano-dispensing Mosquito robot. The crystallization plates are stored in three vibration-free crystallization incubators capable of temperature programming, and crystals are observed using an Olympus SXZ16 stereomicroscope equipped with a 5MP digital camera. Additionally, cryogenic tools, dewars, and a dry shipper from VWR are used for cryo-crystallography and synchrotron data collection. X-ray crystallographic experiments will be performed using a Bruker Microstar high brilliance rotating anode X-ray generator, an Oxford XCalibur sealed tube generator with CCD detector, or a synchrotron. Vanderbilt owns a 1/2 share of the Life Sciences Collaborative Access Team (LS-CAT) beamline which provides over 40 days of synchrotron experiments per year.

NMR spectroscopy. NMR experiments will be conducted in the Center for Structural Biology, which houses one 500, two 600s, one 800, and one 900 MHz Bruker NMR spectrometer as a shared resource. Except for one of the 600s, all spectrometers are equipped with cryoprobes. For NMR-based screening, the 500 MHz and one of the 600 MHz NMR spectrometers are equipped with a Sample Jet sample changer capable of handling batches of up to 480 samples.

Cell Biology. All the equipment necessary to carry out the cell biology experiments is available in our lab or is available for use in the Molecular Biology Core, Cell Biology Core, or High Throughput Screening Core, which occupy lab space immediately adjacent or in close proximity to our group. This equipment includes 4 laminar floor cell culture hoods, 8 temperature controlled incubators (including CO2 regulated incubators and a refrigerated incubator), centrifuges, a Biorad iQ5, a BioRad MYQ5, a BioRad iCycler for Taqman analysis, one LiCor Odyssey, one SpectraMax M5, a Pharos FX Plus Molecular Imager, a Dewar for cryopreservation, a coulter counter, a bioreactor, a microtiter plate reader, an electroporator, and microscopes.

Chemistry. The chemistry portion of the lab consists of 8 fume hoods furnished with the requisite stir plates, manifolds, and glassware to each support a functioning chemist and the equipment necessary to synthesize and purify compounds. This equipment includes:  one  Agilent 6140 Rapid Res analytical LC/ MS system equipped with an additional Varian 380 ELSD detector and Chem-

  Station software; two Gilson preparative HPLC systems equipped with 215 liquid handlers, 845Z prep injection modules, H3 dual piston pumps, and Trilution software; one Biotage Initiator-60 microwave synthesizer; two ISCO combiflash Rf purification systems; four Welch direct drive high vacuum pumps; four IKA RV-10 rotary evaporation systems, complete with Vacubrand PC 3001 pumps; two Glass-Col heated rapid dry-down units; one 20 cu ft Isotemp -20°C freezer; three undercounter VWR refrigerators; two Denver Instruments analytical balances; one Denver Instruments high capacity toploading balance; five Thermo LabQuake rotary shakers; and ventilated chemical storage.

We have a wide selection of polymer supported or resin-bound reagents and scavengers (e.g. PSTrisamine, MP-carbonate, PS-DCC, etc) to enable parallel chemistry, faster reaction workups and purifications. We also have access to an automated mass directed Agilent HPLC system that will be used for the purification of large libraries of compounds. In addition, through the Vanderbilt Institute of Chemical Biology’s Chemical Synthesis Core, the chemistry team has access to additional specialized equipment, including a Varian Polaris 211 analytical HPLC system with PDA and autosampler dedicated to chiral analyses, a Varian Prepstar SD1 preparative scale HPLC dedicated to chiral purifications, three large capacity chiral preparatory columns, a Parr hydrogenator, and an Autopol III automatic polarimeter.Selected additional instrumentation, such as gas chromatographs, an ozonizer, and a high pressure hydrogenation apparatus, among others, is also available at need from the groups of Professors Sulikowski, Lindsley, and Johnston, which are located in close proximity. Within the Mass Spectrometry Core facility, 41 mass spectrometers, including MALDI and TOF instruments, are available for non-routine experiments.

Computing. Each member of the Fesik lab has a Macintosh computer and access to a number of standard computing programs. In addition, three linux work stations are available in the lab for molecular modeling and docking experiments. The lab members have access via subscription to a number of shared software packages, including the Molecular Operating Environment (MOE), the ChemCart database containing the Vanderbilt compound collection, and Pipeline Pilot, via the HTS Core and/or the Center for Structural Biology. High performance and throughput computing are handled by three primary systems. The Advanced Computing Center for Research and Education (ACCRE) maintains a 1200-processor compute cluster with 1.5GB of RAM per processor. The CSB faculty typically access ~15% of ACCRE. The facility is operated by ACCRE staff, who are supported through user fees. The Center for Structural Biology (CSB), of which Dr. Fesik is a member, maintains additional high performance computing resources: an SGI Altix system with 32 Intel Itanium2 CPUs and 64GB of RAM, and a 25-node cluster with 624GB of RAM and 200 Intel Nehalem processor cores interconnected with a 40 Gbit Infiniband fabric. These systems are all interconnected to a 20TB highperformance gigabit Ethernet network-attached storage system which facilitates data sharing between all the above computing resources and local workstations. These CSB facilities are operated by the CSB Computing Core, which is responsible for supporting all local computing resources. This group keeps the central computers and network updated, trouble-shoots, coordinates for outside repair and service contracts, does regular data backups and maintains shared software and databases, remote access into the network, color and black-and-white laser printers, and a data management system for short-term and archival file storage. The CSB Computing Core is supported through a system of user fees. Access to a comprehensive suite of continually updated and optimized structural biology software packages is provided through the SBGrid consortium, which charges an annual fee for this service.