Plant seedlings when inoculated with endophytic diazotrophs showed enhanced growth. Both above ground shoot and below ground roots increased almost 50% relative to uninoculated controls. We were able to locate fluorescence signal from the strain R1Gly cells bearing the reporter gene gfp localized in the certain plant tissue. This indicated that strains were active inside seedlings, and fixing Nitrogen in-planta. In order to identify carbon substrates that potentially attract N2-fixing bacteria to plants, diazotrophic isolates were grown in root exudates collected from hydroponically grown plants, and analyzed for substrate uptake/release. Carbon compounds that are consumed by endophytic bacterial strain and carbon compounds that are excreted by this strain have been identified. The goal of this project is to establish an x-ray footprinting program at the Advanced Light Source. XF is a technique that allows determination of protein structure and dynamics in the solution state at the resolution of a single amino acid. With this technique, the probing reagent – hydroxyl radicals – are produced by energetic photons alone, and no extraneous reagents produce additional unwanted protein modifications. After exposure, samples are digested and analyzed with mass spectrometry to determine which amino acids were modified by the hydroxyl radicals; this results in a map of the solvent-accessible areas of a protein, indicating sites of protein-protein or protein-substrate interactions. Since the necessary exposures are on the order of milliseconds,led grow lights mixing reactions can be performed to give snapshots of protein dynamics or protein-protein interactions as a function of time.
The methodology of data collection, buffer calibration, and mass spectrometry analysis for x-ray foot printing has been previously developed and used very successfully at the NSLS over the last decade. Establishment of an XF program at the Advanced Light Source serves two purposes: 1) it allows continued support of projects currently underway at the NSLS beamline X28C and 2) it further develops and extends the technique of XF into the sub-millisecond time regime.With the previous two years of LDRD funding, an XF program was established at the ALS; experimental feasibility was established using protein and fluorophore standards, and resulted in a publication . Further, results from ALS beamline 5.3.1 demonstrated that foot printing can be conducted in the microsecond domain when using a white-light focusing optic from an ALS bend magnet source, representing a significant extension of the technique. These results were published . An MOU with the ALS Experimental Systems Group was established, allowing access to beamlines 5.3.1 and 3.2.1 on a regular basis. In addition, permission has been obtained from ALS Management to commission beamline 3.3.1 as a dedicated foot printing beamline at the ALS, and initial top-off calculations of the 3.3.1 aperture show that the beamline can be brought online without further shielding work. In addition, we have established an MOU with the NSLS to support NSLS XF users starting in Oct 2014 using beamlines 5.3.1 and 3.2.1 at the ALS. Several important systems were investigated following establishment of the technique at the ALS, and one example is the orange carotenoid protein . In cyanobacteria, this 35 kDa protein is involved in a photoprotective mechanism, undergoing a conformational change under high light conditions that results in a cascade of protein–protein interactions to control the blue green algae photo system. XF experiments on OCP at ALS beamline 5.3.1 showed a significant rearrangement of residues on one side of the carotenoid under blue light illumination.
The results point to an opening of one domain of the protein during light exposure, allowing access to the carotenoid for interaction with other proteins in the complex. This is a significant contribution to understanding the mechanism of activation for this protein, especially given that the “red” state of OCP has been intractable to crystallization. These results have written up and submitted to Nature. The purpose of this project is to understand light-induced water splitting in green plants, algae and cyanobacteria; organisms that are responsible for producing most of the oxygen in the atmosphere. An important application of this knowledge will be in the design of future fuel production schemes based on artificial photosynthesis. The splitting of water, creating oxygen and hydrogen, is accomplished by the protein complex photo system II, which contains a catalytic center containing four manganese atoms. To drive the reaction to completion, four sunlight photons sequentially oxidize the Mn atoms after which the catalyst returns to the reduced state. Traditional methods for studying structure and function, such as X-ray crystallography, have been hampered by the high sensitivity of the Mn center to probing X-rays, which reduce the metal atoms to the Mn valence state. We have an unprecedented opportunity to map out the detailed reaction mechanism using X-ray free-electron laser experiments at the Linac Coherent Light Source .X-ray probe pulses at LCLS are short enough that all observations can be made before reduction and other damage processes occur.
As the probe pulses are extremely intense, the sample becomes fully ionized after the observations are recorded; therefore, a continuous stream of new crystals is required for a full data set. Atomic structure of the protein will be probed by X-ray diffraction, while simultaneously the electronic state of the Mn atoms will be measured by X-ray emission and spectroscopy. The experimental team is a collaboration between many groups at different institutions. This LDRD is specifically targeted at developing the requisite computational methods to handle the large data sizes, with the involvement of NERSC resources. In it’s third and final year , the project published XFEL-derived crystal structures of photosystem II in the dark-equilibrated state, as well as in three illuminated states along the reaction coordinate. While our published structural data extends to 4.5 Å resolution only, our latest experiments with optimized crystal growth conditions indicate that 2.5 Å data are achievable. We published extensive computational methods for XFEL diffraction experiments applied to structural biology. The unique data collection conditions required us to develop analysis methods to optimally model the crystal orientation and internal physical properties. Our software is general and widely applicable to XFEL-based protein crystallography. In collaboration with Axel Brunger we created procedures for organizing large datasets where the diffraction quality is heterogeneous, explored systematic correction methods for converting the Bragg spots to reduced Fourier coefficients for calculating the electron density map, and contributed to the development of a new microfluidic trap device that will hopefully reduce the amount of sample required for a full dataset. With David Eisenberg we developed new algorithms to process very sparse crystal diffraction patterns from small molecules. We’ve used XFEL diffraction to investigate small peptide fragments that determine the β–sheet protein structure in numerous amyloid diseases,vertical grow system and in particular the mechanism of a single-amino acid mutation causing Parkinson’s disease. As data sets grow larger, there is an increasing need across multiple LBNL divisions for tools to simplify “High Throughput Computing” — the efficient processing of many thousands or even millions of small independent tasks. For example, users need to apply the same analysis program to many thousands of images or spectra or gene sequences, or run the same simulation with thousands of different input parameters. The traditional batch model of 1 task = 1 script = 1 job becomes impractical for both the batch queue system and the human management of such scripts. At the same time, pre-bundling multiple tasks into a single batch script lacks flexibility, e.g. for processing new data as it arrives. We are developing an alternate model to simplify HTC workflows at NERSC and elsewhere, based upon separating the queue of tasks to perform from the batch jobs that process those tasks.
This work will simplify HTC workflows at NERSC, attract new users, and reduce the software development costs for future projects with big data HTC processing requirements. In the first year of the project we submitted a wafer design to Teledyne DALSA, and the CCD wafer fabrication was successful. The second year of the project described here utilized these wafers in the MSL for QE improvement development and for testing the low noise CCD designs. The figure below shows the improvements in QE at both blue and red wavelengths that have been demonstrated. The measured QE on recently-fabricated CCDs is shown by the red symbols, and that is compared to the standard QE shown in the blue symbols. The improvements are due to the use of a thinner backside contact layer and the use of improved antireflection coating materials, specifically the use of ZrO2 in the AR coating. We also demonstrated improved noise performance. In addition to the use of a direct connection between the CCD sense node and output transistor polysilicon gate electrode that we had described previously, we also explored the use of a thinner gate-insulator layer in the output transistor. This allowed for the use of a smaller gate length in the output transistor, and resulted in a reduction of the noise floor from about 1 e- rms to about 0.8 e- rms. We also explored the use of mixand-match lithography to reduce the size of the sense node to 1 × 1 µm2 from 2 × 2 µm2 . Although this did not result in a noticeable noise improvement, the ability to utilize the finer-line lithography could be useful for future noise improvements. These enhancements were included on both small format CCDs, and on 16- channel CCDs to allow for improved statistics. See the listed publication for more details. We have also made progress on single-photon detection via impact ionization in a charge multiplying CCD, with the first demonstration for us of the multiplication of sub-electron light levels with realistic gain values in our p-channel technology. A first prototype chip was designed in FY12 and fabricated in FY13. This chip contains an active pixel matrix. Working devices were received in February 2013 and then tested to validate the design methods used and simulation results. These tests were successful, leading to submission and presentation of results at the International Image Sensor Workshop in June 2013. Following this, devices were irradiated in September wiht protons to a maximum dose of 30Mrad. Testing of the irradiated devices took place in FY14. In FY14 devices were also characterized for minimum ionizing particle detection efficiency using the SLAC test beam facility. The conclusion from these studies is that the concept works, but cannot achieve the desired 99% detection efficiency for MIP detection. Instead, the MIP efficiency of the tested devices is between 50% and 60%. Irradiaiton did not degrade the performance. To increase efficiency one must reduce capacitance and/or increase the collected charge. A test structure chip was fabricated in FY14 to explore the process parameter space for the charge collection implant to minimize capacitance and maximize breakdown voltage. This characterization was successful, but the results disappointing, showing very little room for improvement. Finally, in FY14, a redesign of the pixel geometry was carried out to reduce the size and therefore the capacitance of the pixel. However, this design was not fabricated. Even with these improvements, simulations show that the efficiency would reach just 80%. A higher substrate resistivity is needed to boost the signal and reach the desired efficiency . At the conclusion of the LDRD no higher resitivity process with the needed deep implant feature was available. The purpose of this project is to develop detector technology for next-generation Cosmic Microwave Background polarimetry experiments. Primordial gravitational waves produced during inflation and gravitational lensing by large-scale structure produce “B-mode” polarization patterns on the sky which have a handedness. A detection of primordial B-modes would be a “smoking gun” for inflation as the origin of the universe and would determine inflation’s energy scale. A detailed characterization of the lensed B-modes would also allow us to constrain the sum of the neutrino masses and the evolution of dark energy by cross-correlating with BAO experiments such as DESI. Just last year, experiments such as POLARBEAR detected B-modes from gravitational lensing directly for the first time. Current CMB experiments are deploying of order 1,000 detectors and experiments with order 10,000 detectors will deploy within few years. The community is discussing a possibility of stage-four CMB experiment to definitively characterize B-mode polarization with a order 500,000 detector count.