Technologies Developed at the ATCG3D
Synthetic Gene Design and Whole Gene Synthesis
Name: Gene Composer™ Software for Synthetic Gene Design and Construct Engineering
Summary: Gene Composer™ software for computer aided construct engineering and expression optimized synthetic gene design is available for download from www.genecomposer.net.
Description: Gene Composer™ software for computer aided construct engineering and expression optimized synthetic gene design are available for download from www.genecomposer.net. Gene Composer™ is a complete synthetic gene design suite for Microsoft Windows® which runs on top of a single user Access® database with an Alignment Viewer, Construct Design Module, and Gene Design Module. In addition, the Virtual Cloning Module features the ability to permute gene constructs into defined expression vectors by way of user defined adaptor assemblies that recapitulate most wet lab cloning procedures. The Gene Composer™ Protein-to-DNA algorithm allows users to design codon optimized DNA sequences based on the original cDNA or amino acid sequence data, while introducing or eliminating other defined DNA sequence elements. For example, all ACA sequences can be silently removed from open reading frames, so that they may be used in conjunction with the MazF system for single protein production.
ATCG3D has hosted on-line workshops on the use of Gene Composer™ software for expression optimized synthetic gene design and protocols for PCR-based gene synthesis from designed oligonucleotides and improved error reduction through the use of mis- match specific endonucleases. Workshop video recordings can be requested at www.genecomposer.net.
An example of the use of synthetic gene design by Gene Composer™ is reported in DcpS as a therapeutic target for spinal muscular atrophy (2008) ACS Chemical Biology, 3, 711-722 (PubMed ID: 18839960).
This paper demonstrates the importance of protein structures in translational medicine. It describes the identification of human DcpS (mRNA decapping scavenger enzyme) as a therapeutic target for spinal muscular atrophy (SMA). A series of C-5 substituted quinazoline molecules which inhibit DcpS were shown to enhance expression of the SMN2 gene whose improved expression may help patients with SMA. To facilitate this discovery, ATCG3D researchers synthetically engineered a novel DcpS gene for protein expression and crystallization with the aid of Gene Composer which was developed with ATCG3D funding (www.genecomposer.net). ATCG3D researchers went on to solve the high resolution X-ray crystal structures of DcpS bound to several of the most active C5-substituted quinazolines.
Emerald BioSystems is responsible for commercialization of Gene Composer™ (www.emeraldbiosystems.com).
Contact: Lance Stewart, lstewart@embios.com
________________________________________________________________Top of the page
Nanovolume Microfluidic Crystallization in Confined Geometries
Name: Nanovolume plug-based microfluidic crystallization in CrystalCards™
Summary: The Microcapillary Protein Crystallization System (MPCS™) utilizes CrystalCards™, plastic microfluidic devices designed to prepare and store approximately 800 individual nanovolume crystallization experiments that produce Diffraction-Ready™ protein crystals. The peel-apart nature of the CrystalCards allows the researcher to remove crystals from the microfluidic device for X-ray diffraction data collection using standard laboratory equipment and techniques. The CrystalCards are also sufficiently X-ray transmissive to allow in situ X-ray diffraction data collection from crystals held in devices placed directly within the X-ray beam.
Description: Originally developed by the Ismagilov lab at the University of Chicago, nanovolume plug-based microfluidic protein crystallization is an emerging technology for efficient protein use for crystallization trials in conjunction with in situ X-ray imaging. Emerald and its microfulidic device collaborators have produced several different plastic labcards that contain of the required microfluidic circuitry to prepare several hundred nanovolume (~20 nl) protein crystallization experiments as aqueous plugs that are carried in a microfluidic channel by an inert fluoropolymeric oil (fluorinert). Three aqueous microfluidic channels for protein, buffer and precipitant converge in a mixer circuit together with the fluorinert carrier fluid (a 3+1 Mixer). Microsyringe pumps controlled by Microplugger™ software drive the formation of nanovolume aqueous plugs into the fluorinert carrier fluid which enters a long microfluidic holding circuit where final nanovolume crystallization experiments are stored.
Functional 3+1 Mixer + channel holder CrystalCards made out of cyclic olefin copolymer (COC) and polycarbonate (PC) material have been produced with chemical treatments that lower the surface energy of the microfluidic circuitry, making it functional for nanovolume aqueous plug formation. The labcards have proved to be of reasonable reliability in running gradient method nanovolume plug-based protein crystallizations using test proteins (lysozyme and thaumatin) as well as other experimental proteins. In situ X-ray diffraction on in-house sources have been demonstrated for both COC and PC types of labcards with reasonable success. Emerald BioSystems is responsible for commercialization of the microfluidic protein crystallization crystallization system (www.emeraldbiosystems.com).
The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS). Acta Crystallographica Section D: Biological Crystallography, 64, 1116-1122 (PubMed ID: 19020349).
Contact: Lance Stewart, lstewart@embios.com
Availability: Microcapillary Protein Crystallization System™ (MPCS™)
MPCS™ Features
MPCS™ Plug Maker™
Name: A Hybrid Method for Plug-Based Microfluidic Protein Crystallization
Summary: A microfluidic “hybrid” method for plug-based microcapillary protein crystallization has been developed to combine sparse-matrix and optimization-gradient screening into one simple experiment to minimize sample consumption.
Description: ATCG3D developed a microfluidic “hybrid” method that combines sparse-matrix and optimization-gradient screening into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption (PNAS (2006) 103: 19243-19248). Many distinct reagents were sequentially introduced as large plugs (~ 140 nL) into a microfluidic device. Large plugs were combined with a protein sample and a diluting buffer so that multiple ~10 nL plugs of each reagent were formed over a range of concentrations. The concentrations of each reagent were well-controlled by a computer subroutine and were indexed with plug size. We validated the hybrid method by demonstrating its applicability to two challenging problems: i) crystallization of model membrane proteins and ii) handling of detergent solublized membrane protein and viscous precipitants. We overcome these challenges with two technical developments: i) the use of perfluoroamines as carrier fluids, and ii) the use of Teflon capillaries for the formation, transport, and storage of plugs. We obtained high-quality crystals of membrane proteins and solved a crystal structure. This robust method requires inexpensive equipment and supplies, is accessible to most basic individual laboratories, and could find applications in a number of areas that require chemical, biochemical or biological screening and optimization.
Proceedings of the National Academy of Sciences of the United States of America, 103 (51), 19243-19248 (PubMed ID: 17159147).
Contact: Rustem Ismagilov, rustem@uchicago.edu
Name: In situ X-ray analysis of protein crystals in low-birefringent and X-ray transmissive plastic microchannels
Summary: Very small volumes of protein sample can be used in crystallization regimes of restricted geometry including microfluidic channels. Crystal to crystal diffraction quality differences can be revealed through in situ X-ray diffraction analysis of crystals grown in microfluidic restricted geometry microchannels.
Description: Plastic microchannel crystallization template designs made from inexpensive cyclic olefin copolymers have been shown to be low-birefringent, X-ray transmissive and compatible with microfluidic fabrication in restricted geometry. The model proteins thaumatin, lysozyme and bacteriorhodopsin demonstrated the feasibility of conducting counter-diffusion equilibration within the new plastic configuration. Crystals of each of these proteins were directly evaluated in situ using synchrotron radiation and their diffraction quality was evaluated without invasive manipulation or cryofreezing. Protein crystals able to produce complete X-ray data sets were used to calculate electron-density maps for structure determination. Fluidic crystallization in the plastic platform was also coupled with a commercialized automated imager and an in situ X-ray scanner that allowed optical and X-ray inspection of crystallization hits. The results demonstrate the feasibility of rapid nanovolume counter-diffusion crystallization experiments without the need for additional instrumentation.
Acta Crystallographica Section D: Biological Crystallography, 64, 189-197 (PubMed ID: 18219119) and Methods in Molecular Biology, 426, 363-376 (PubMed ID: 18542876).
Contact: Peter Kuhn, pkuhn@scripps.edu
Name: Plug-Based Microfluidic Seeding Method for Protein Crystallization
Description: We have developed a microfluidic method of seeding to separate and independently control the stages of nucleation and growth in protein crystallization (Angew. Chem. Int. Ed. 2006 45: 8156-8160). This seeding method enables the generation of high quality protein crystals in nanoliter volumes with minimal sample consumption, and it is especially suitable for crystallizing proteins that display a supersaturation gap on their phase diagram. In a typical experiment, seeds are first generated in plugs under high supersaturation conditions in the nucleation stage. Next, these seeds are merged with aqueous plugs at lower supersaturation to initiate the growth stage and allow formation of ordered crystals. This method was validated by growing crystals of Oligoendopeptidase F. The crystal structure of Oligoendopeptidease F was solved at 3.3 Ǻ.
Contact: Rustem Ismagilov, rustem@uchicago.edu
Name: The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist
Summary: This paper reports the second human GPCR structure to be resolved by X-ray crystallography using a combination of T4-Lysozyme fusion and lipid mesophase technologies.
Description: The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.
The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science, 322, 1211-1217 (PubMed ID: 18832607).
Contact: Raymond Stevens, stevens@scripps.edu
Name: C-ME: a 3D community-based, real-time collaboration tool for scientific research and training
Summary: C-ME is a collaborative web-enabled community-based database software application for 3D protein structure annotation.
Description: The need for effective collaboration tools is growing as multidisciplinary proteome-wide projects and distributed research teams become more common. The resulting data is often quite disparate, stored in separate locations, and not contextually related. Collaborative Molecular Modeling Environment (C-ME) is an interactive community-based collaboration system that allows researchers to organize information, visualize data on a two-dimensional (2-D) or three-dimensional (3-D) basis, and share and manage that information with collaborators in real time. C-ME stores the information in industry-standard databases that are immediately accessible by appropriate permission within the computer network directory service or anonymously across the internet through the C-ME application or through a web browser. The system addresses two important aspects of collaboration: context and information management. C-ME allows a researcher to use a 3-D atomic structure model or a 2-D image as a contextual basis on which to attach and share annotations to specific atoms or molecules or to specific regions of a 2-D image. These annotations provide additional information about the atomic structure or image data that can then be evaluated, amended or added to by other project members.
PLoS ONE, 3, e1621 (PubMed ID: 18286178).
Contact: Peter Kuhn, pkuhn@scripps.edu
Name: DETECT-X Crystal Imaging Technology
Summary: The DETECT-X (Difference Extinction for the Detection of Crystals) microscope system and image processing algorithms allow for improved protein crystal imaging.
Description: We have developed a DETECT-X (Difference Extinction for the Detection of Crystals) microscope system and associated image processing algorithms for improved crystal imaging. The DETECT-X microscope is a fully automated polarization and UV fluorescence microscope for inspecting crystallization trials. Through the analysis of four polarization images (at 0, 45, 90, 135) the system calculates quantitative values for the transmission, the birefringence, and the extinction angle at each pixel. Each of these values can then be displayed as a false-color image and together these images provide better contrast for identifying crystalline material. In its birefringence microscopy mode the DETECT-X microscope produces quantitative images of crystals depicting the orientation of the slow optical axis of protein crystals and their orientation-independent, quantitative birefringence. Such images allow the detection of crystals that are masked by precipitate. Spherulites and microcrystalline material can be readily distinguished from amorphous precipitate. As a result, there are fewer false negatives and more potential leads are identified for crystal optimization. Twinned crystals can also be readily identified, so that the best crystals for diffraction experiments can be selected. The use of in situ UV fluorescence microscopy also helps to distinguish between salt crystals and protein crystals. It is anticipated that DETECT-X microcopy improved efficiencies in optical screening of protein crystallization trials.
Contact: Peter Nollert, pnollert@embios.com
Avaliabilty: DETECT-X User Login
Name: Simple host-guest chemistry to modulate the process of concentration and crystallization of membrane proteins by detergent capture in a microfluidic device
Summary: This paper describes detergent titration by cyclodextrins in a microfluidic plug-based mode for crystallization of membrane proteins. The cyclodextrin titration can be applied to reduce free micelle concentrations both during crystallization and also during concentration of membrane protein samples, both of which are generally applicable to membrane protein crystallization.
Description: This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-beta-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.
Simple host-guest chemistry to modulate the process of concentration and crystallization of membrane proteins by detergent capture in a microfluidic device. Journal of the American Chemical Society, 130, 14324-14328 (PubMed ID: 18831551).
Contact: Rustem Ismagilov, rustem@uchicago.edu
________________________________________________________________Top of the page
Compact Light Source
Name: The ATCG3D Compact Light Source
Summary: ATCG3D is funding the construction of a tunable laboratory X-ray source called the Compact Light Source for protein X-ray crystallography applications (www.lynceantech.com).
Description: The Compact Light Source is a miniature room-sized synchrotron light source that will enable scientists in academics and industry to pursue state-of-the-art synchrotron radiation applications in their own laboratories. The Compact Light Source, will deliver a monochromatic pencil beam of tunable hard X-rays for both medical imaging applications and protein X-ray crystal structure determination. The X-ray beam energy can be tuned from a few keV up to 35 keV.
Contact: Ronald Ruth, ronald_ruth@lynceantech.com
________________________________________________________________Top of the page