Engineering topics

vanderWoerd, Mark

Publications and source records attributed to vanderWoerd, Mark.

12 records

NASA NTRS2006

Thermal Imaging Applied to Cryocrystallography: Cryocooling and Beam Heating (Part I)

Thermal imaging provides a non-invasive method to study both the cryocooling process and the heating due to the X-ray beam interaction with a sample. The method has been used successfully to image cryocooling in a number of experimental situations, i.e. cooling as a function of sample volume and as a function of cryostream orientation. Although there are experimental limitations to the method, it has proved a powerful technique to aid cryocrystallography development. Due to the rapid spatial temperature information provided about the sample it is also a powerful tool in the testing of mathematical models. Recently thermal imaging has been used to measure the temperature distribution on both a model and typical crystal samples illuminated with an X-ray beam produced by an undulator. A brief overview of thermal imaging and previous results will be presented. In addition, a detailed description of the calibration and experimental aspects of the beam heating measurements will be described. This will complement the following talk on the mathematical modeling and analysis of the results.

Snell, Edward↗

NASA NTRS2006

Beam Heating of Samples: Modeling and Verification

Energy absorbed from the X-ray beam by the sample requires cooling by forced convection (i.e. cryostream) to minimize temperature increase and the damage caused to the sample by the X-ray heating. In this presentation we will first review the current theoretical models and recent studies in the literature, which predict the sample temperature rise for a given set of beam parameters. It should be noted that a common weakness of these previous studies is that none of them provide actual experimental confirmation. This situation is now remedied in our investigation where the problem of x-ray sample heating is taken up once more. We have theoretically investigated, and at the same time, in addition to the numerical computations, performed experiments to validate the predictions. We have modeled, analyzed and experimentally tested the temperature rise of a 1 mm diameter glass sphere (sample surrogate) exposed to an intense synchrotron X-ray beam, while it is being cooled in a uniform flow of nitrogen gas. The heat transfer, including external convection and internal heat conduction was theoretically modeled using CFD to predict the temperature variation in the sphere during cooling and while it was subjected to an undulator (ID sector 19) X-ray beam at the APS. The surface temperature of the sphere during the X-ray beam heating was measured using the infrared camera measurement technique described in a previous talk. The temperatures from the numerical predictions and experimental measurements are compared and discussed. Additional results are reported for the two different sphere sizes and for two different supporting pin orientations.

Kazmierczak, Michael↗

NASA NTRS2006

Optimizing Crystal Volume for Neutron Diffraction

Neutron diffraction is uniquely sensitive to hydrogen positions and protonation state. In that context structural information from neutron data is complementary to that provided through X-ray diffraction. However, there are practical obstacles to overcome in fully exploiting the potential of neutron diffraction, Le. low flux and weak scattering. Several approaches are available to overcome these obstacles and we have investigated the simplest: increasing the diffracting volume of the crystals. Volume is a quantifiable metric that is well suited for experiment design and optimization techniques. By using response surface methods we have optimized xylose isomerase crystal volume, enabling neutron diffraction while we determined the crystallization parameters with the minimum of experiments. Our results suggest a systematic means of enabling neutron diffraction studies for a larger number of samples that require information on hydrogen position and/or protonation state.

Snell, Edward H.↗

NASA NTRS2006

A Quasi-Laue Neutron Crystallographic Study of D-Xylose Isomerase

Hydrogen atom location and hydrogen bonding interaction determination are often critical to explain enzymatic mechanism. Whilst it is difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (less than 1.0 Angstrom) resolution data available, neutron crystallography provides an experimental tool to directly localise hydrogeddeuteriwn atoms in biological macromolecules at resolution of 1.5-2.0 Angstroms. Linearisation and isomerisation of xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data were collected on Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism (Myles et al. 1998). The neutron structure unambiguously reveals the protonation state of His 53 in the active site, identifying the model for the enzymatic pathway.

Meilleur, Flora↗

NASA NTRS2004

DNA in a Tunnel: A Comfy Spot for Recognition - or -The Structure of BsoBI Complexed with DNA. What can we Learn about Function via Structure Determination and how can this be Applied to Bone or Muscle Biology?

The structure and function of a biologically active molecule are related. To understand its function, it is necessary (but not always sufficient) to know the structure of the molecule. There are many ways of relating the molecular function with the structure. Mutation analysis can identify pertinent amino acids of an enzyme, or alternatively structure comparison of the of two similar molecules with different function may lead to understanding which parts are responsible for a functional aspect, or a series of "structural cartoons" - enzyme structure, enzyme plus substrate, enzyme with transition state analog, and enzyme with product - may give insight in the function of a molecule. As an example we will discuss the structure and function of the restriction enzyme BsoBI from Bacillus stearothemzophilus in complex with its cognate DNA. The enzyme forms a unique complex with DNA in that it completely encircles the DNA. The structure reveals the enzyme-DNA contacts, how the DNA is distorted compared with the canonical forms, and elegantly shows how two distinct DNA sequences can be recognized with the same efficiency. Based on the structure we may also propose a hypothesis how the enzymatic mechanism works. The knowledge gained thru studies such as this one can be used to alter the function by changing the molecular structure. Usually this is done by design of inhibitors specifically active against and fitting into an active site of the enzyme of choice. In the case of BsoBI one of the objectives of the study was to alter the enzyme specificity. In bone biology there are many candidates available for molecular study in order to explain, alter, or (temporarily) suspend activity. For example, the understanding of a pathway that negatively regulates bone formation may be a good target for drug design to stimulate bone formation and have good potential as the basis for new countermeasures against bone loss. In principle the same approach may aid muscle atrophy, radiation damage, immune response changes and other risks identified for long-duration Space travel.

vanderWoerd, Mark↗

NASA NTRS2003

Protein Crystal Growth With the Aid of Microfluidics

Protein crystallography is one of three well-known methods to obtain the structure of proteins. A major rate limiting step in protein crystallography is protein crystal nucleation and growth, which is still largely a process conducted by trial-and-error methods. Many attempts have been made to improve protein crystal growth by performing growth in microgravity. Although the use of microgravity appears to improve crystal quality in some attempts, this method has been inefficient because several reasons: we lack a fundamental understanding of macromolecular crystal growth in general and of the influence of microgravity in particular, we have to start with crystal growth conditions in microgravity based on conditions on the ground and finally the hardware does not allow for experimental iteration without reloading samples on the ground. To partially accommodate the disadvantages of the current hardware, we have used microfluidic technology (Lab-on-a-Chip devices) to design the concept of a more efficient crystallization device, suitable for use on the International Space Station and in high-throughput applications on the ground. The concept and properties of microfluidics, the application design process, and the advances in protein crystal growth hardware will be discussed in this presentation. Some examples of proteins crystallized in the new hardware will be discussed, including the differences between conventional crystallization versus crystallization in microfluidics.

vanderWoerd, Mark↗

NASA NTRS2003

The Promise of Macromolecular Crystallization in Micro-fluidic Chips

Micro-fluidics, or lab on a chip technology, is proving to be a powerful, rapid, and efficient approach to a wide variety of bio-analytical and microscale bio-preparative needs. The low materials consumption, combined with the potential for packing a large number of experiments in a few cubic centimeters, makes it an attractive technique for both initial screening and subsequent optimization of macromolecular crystallization conditions. Screening operations, which require equilibrating macromolecule solution with a standard set of premixed solutions, are relatively straightforward and have been successfully demonstrated in a micro-fluidics platform. More complex optimization methods, where crystallization solutions are independently formulated from a range of stock solutions, are considerably more complex and have yet to be demonstrated. To be competitive with either approach, a micro-fluidics system must offer ease of operation, be able to maintain a sealed environment over several weeks to months, and give ready access for the observation of crystals as they are grown.

vanderWoerd, Mark↗

NASA NTRS2003

Finding the Cold Needle in a Warm Haystack: Infrared Imaging Applied to Locating Cryo-cooled Crystals in Loops

Thermally imaging the cryocooling processes of crystals has been demonstrated showing the progression of a cold wave through a crystal from the face closest to the origin of the coldstream ending at the point furthest away. During these studies large volume crystals were clearly distinguished from the loop holding them. Large volume crystals, used for neutron studies, were chosen deliberately to enhance the imaging. The different infrared transmission and reflectance properties of the crystal in comparison to the cryo-protectant are thought to be the parameter that produces the contrast making the crystal visible. As an application of the technology to locating crystals, more small crystals of lysozyme and a bFGF/dna complex were cryo-protected and imaged in large loops. The crystals were clearly distinguished from the vitrified solution. In the case of the bFGF/dna complex the illumination had to be carefully manipulated to enable the crystal to be seen in the visible spectrum. These preliminary results will be presented along with advantages and disadvantages of the technique and a discussion of how it might be applied.

Snell, Edward↗

NASA NTRS2003

Perfectly Cold Crystals: What Happens When They Are X-rayed?

For many macromolecular crystals the cryo-preservation of these crystals during X-ray data collection is of crucial importance, particularly at synchrotron facilities where the crystals rapidly receive a high dose of radiation. A practical variable to ensure adequate preservation is the variation of the cryo-protectant present when the crystal is preserved. Our initial approach to study X-ray diffraction data quality as a function of cryo-protectant present when preserving a xylose isomerase crystal shows that the data quality can be tremendously improved by recipe adjustment. Guided by crystal mosaicity estimates, we optimized crystal growth conditions to obtain cryo-preserved xylose isomerase crystals that withstand a very high dose of X-rays, with only the smallest amount of radiation damage at ultra-high resolution (1.2Angstroms). The rate at which damage occurs allowed us to collect a series of complete data sets, which show how the data degradation proceeds over time. We are here presenting data for the xylose isomerase crystallization recipe improvement and our interpretation of the crystal degradation process during X-ray data collection.

vanderWoerd, Mark↗

NASA NTRS2002

About Small Streams and Shiny Rocks: Macromolecular Crystal Growth in Microfluidics

We are developing a novel technique with which we have grown diffraction quality protein crystals in very small volumes, utilizing chip-based, microfluidic ("LabChip") technology. With this technology volumes smaller than achievable with any laboratory pipette can be dispensed with high accuracy. We have performed a feasibility study in which we crystallized several proteins with the aid of a LabChip device. The protein crystals are of excellent quality as shown by X-ray diffraction. The advantages of this new technology include improved accuracy of dispensing for small volumes, complete mixing of solution constituents without bubble formation, highly repeatable recipe and growth condition replication, and easy automation of the method. We have designed a first LabChip device specifically for protein crystallization in batch mode and can reliably dispense and mix from a range of solution constituents. We are currently testing this design. Upon completion additional crystallization techniques, such as vapor diffusion and liquid-liquid diffusion will be accommodated. Macromolecular crystallization using microfluidic technology is envisioned as a fully automated system, which will use the 'tele-science' concept of remote operation and will be developed into a research facility aboard the International Space Station.

vanderWoerd, Mark↗

NASA NTRS2002

Decades of Data: Extracting Trends from Microgravity Crystallization History

The reduced acceleration environment of an orbiting spacecraft has been posited as an ideal environment for biological crystal growth since buoyancy driven convection and sedimentation are greatly reduced. Since the first sounding rocket flight in 1981 many crystallization experiments have flown with some showing improvement and others not. To further explore macromolecule crystal improvement in microgravity we have accumulated data from published reports and reports submitted by individual investigators to NASA, forming a database called BIOSEArCH (Biological Space Experiment Archive of Crystallization History). To date it contains information from 63 missions including, the Space Shuttle program, unmanned satellites, the Russian Space Station MIR and sounding rocket experiments, containing reports for more than 736 macromolecule experiments. While it is not at this point in time a comprehensive record of all flight crystallization experimental results, there is however sufficient information for emerging trends to be identified. These trends will be highlighted.

Judge, Russell A.↗

NASA NTRS2002

Macromolecular Crystal Growth by Means of Microfluidics

We have performed a feasibility study in which we show that chip-based, microfluidic (LabChip(TM)) technology is suitable for protein crystal growth. This technology allows for accurate and reliable dispensing and mixing of very small volumes while minimizing bubble formation in the crystallization mixture. The amount of (protein) solution remaining after completion of an experiment is minimal, which makes this technique efficient and attractive for use with proteins, which are difficult or expensive to obtain. The nature of LabChip(TM) technology renders it highly amenable to automation. Protein crystals obtained in our initial feasibility studies were of excellent quality as determined by X-ray diffraction. Subsequent to the feasibility study, we designed and produced the first LabChip(TM) device specifically for protein crystallization in batch mode. It can reliably dispense and mix from a range of solution constituents into two independent growth wells. We are currently testing this design to prove its efficacy for protein crystallization optimization experiments. In the near future we will expand our design to incorporate up to 10 growth wells per LabChip(TM) device. Upon completion, additional crystallization techniques such as vapor diffusion and liquid-liquid diffusion will be accommodated. Macromolecular crystallization using microfluidic technology is envisioned as a fully automated system, which will use the 'tele-science' concept of remote operation and will be developed into a research facility for the International Space Station as well as on the ground.

vanderWoerd, Mark↗