This proposal requests funds to purchase a robot to set up crystallization experiments for the Structural Biology Resource Center at The Rockefeller University. The robot will aid researchers in obtaining diffraction quality crystals of biological macromolecules involved in bacterial transcription and its regulation, bacterial two-component histidine kinase signaling, bacterial pathogenesis, hepatitis C virus replication, and eukaryotic cell division. Obtaining crystals is often the rate-limiting step in X-ray structure determination. The robot will greatly increase the efficiency of crystallization trials by reducing the amount of time and sample required. This, in turn, will increase the success rate of NIH-funded research, and will increase utilization of the NIH-funded X-ray equipment already in the Resource Center. Due to the smaller quantities of materials required for the robot as opposed to manual setup, the robot will also allow extensive crystallization trials for projects that would otherwise not be possible. In addition, crystallization experiments will have a smaller impact on the environment by generating much less waste. The crystallization robot requested is the Screenmaker96+8 from Innovadyne - $145000. This robot can perform three distinct processes needed to set up crystallization experiments. Step 1: transfer of reservoir solutions from the sparse matrix stocks to the 96 positions in the crystallization tray;Step 2: deposition of nano-liter volumes of this reservoir onto the drop position and deposition of the protein nano-liter volume onto the same spot;Step 3: to refine the condition by making custom grids by varying two components in the lead condition to create new opportunities for improved crystal growth. Another very important feature of the Screenmaker96+8 is that the time required to place the drops (both the precipitant solution and the protein) is significantly less than other crystallization robots, thereby reducing dehydration time by at least half. The crystallization robot will be housed in the newly created Structural Biology Resource Center, supported by The Rockefeller University under the umbrella of the Research Support office. The Center will supervise the robot's use, train users and maintain the instrument in top working condition, and will be responsible for fair, relevant and efficient utilization of the robot. Additional parts needed: computer $2450, installation $4000, two nest chiller system $10450, for sensitive samples and extended warranty $32000. (Prices quoted here do not reflect 10% academic discount applied to total requested in budget, see price quote in Equipment.doc).
Determining the three-dimensional structure of biological macromolecules is key in understanding their function and in opening new venues for designing drugs and cures for human diseases. This can be accomplished through the technique known as X-ray crystallography, where the rate-limiting step is often the crystallization of the target molecule, a menial process that is labor intensive and time consuming. This proposal requests a robot that performs the steps involved in setting up crystallization trials of biologically important molecules, decreasing the labor 10 fold and decreasing the generation of waste while increasing the efficiency and scope of the experimental projects.
|Hubin, Elizabeth A; Lilic, Mirjana; Darst, Seth A et al. (2017) Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures. Nat Commun 8:16072|
|Feklistov, Andrey; Bae, Brian; Hauver, Jesse et al. (2017) RNA polymerase motions during promoter melting. Science 356:863-866|
|Hubin, Elizabeth A; Fay, Allison; Xu, Catherine et al. (2017) Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA. Elife 6:|
|Zamolodchikov, Daria; Berk-Rauch, Hanna E; Oren, Deena A et al. (2016) Biochemical and structural analysis of the interaction between ?-amyloid and fibrinogen. Blood 128:1144-51|
|Singh, Nimisha; Blobel, Günter; Shi, Hang (2015) Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport. Proc Natl Acad Sci U S A 112:142-7|
|Bae, Brian; Chen, James; Davis, Elizabeth et al. (2015) CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex. Elife 4:|
|Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka et al. (2015) Structure of a bacterial RNA polymerase holoenzyme open promoter complex. Elife 4:|
|Hubin, Elizabeth A; Tabib-Salazar, Aline; Humphrey, Laurence J et al. (2015) Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA. Proc Natl Acad Sci U S A 112:7171-6|
|Whorton, Matthew R; MacKinnon, Roderick (2013) X-ray structure of the mammalian GIRK2-?? G-protein complex. Nature 498:190-7|
|Elsässer, Simon J; Huang, Hongda; Lewis, Peter W et al. (2012) DAXX envelops a histone H3.3-H4 dimer for H3.3-specific recognition. Nature 491:560-5|