Results from the Antarctic Muon and Neutrino Detector Array (AMANDA)

We show new results from both the older and newer incarnations of AMANDA (AMANDA-B10 and AMANDA-II, respectively). These results demonstrate that AMANDA is a functioning, multipurpose detector with significant physics and astrophysics reach. They include a new higher-statistics measurement of the atmospheric muon neutrino flux and preliminary results from searches for a variety of sources of ultrahigh energy neutrinos: generic point sources, gamma-ray bursters and diffuse sources producing muons in the detector, and diffuse sources producing electromagnetic or hadronic showers in or near the detector.Comment: Invited talk at the XXth International Conference on Neutrino Physics and Astrophysics (Neutrino 2002), Munich, Germany, May 25-30, 200

A linear relationship between crystal size and fragment binding time observed crystallographically: implications for fragment library screening using acoustic droplet ejection.

High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size

Crystallization, data collection, and model refinement statistics.

<p>X-ray diffraction data sets were obtained from 354 lysozyme crystals soaked with N-acetyl glucosamine and from 103 thermolysin crystals soaked with asparagine. Each X-ray data set was used to estimate the refined occupancy of the ligand (O_refine) and a least squares procedure was used to fit Eq. 1 to these occupancies. The two fitted parameters were the occupancy at infinite time (O_max, from which an intra-crystalline dissociation constant K_d^cryst can be calculated using Eq. 2) and a fitting parameter related to diffusion speed (τ). For each measured value “x”, both the mean “x¯” and the population standard deviation “σ(x)” are listed as x¯ ± σ(x). The population standard deviation was calculated using the formula σ(x) = (Σ (x−x¯)²/n)^½, where “n” is the number of measurements (always equal to 354 for lysozyme data and 103 for thermolysin data).</p

Precision and accuracy of the occupancy calculation and the K_d^cryst value.

<p>X-ray diffraction data were obtained from 10 thermolysin crystals that were not soaked in asparagine (first column) and from 10 thermolysin crystals that were soaked in 100 mM asparagine overnight (second column). We disregard crystal size because of the long soak times (all crystals were approximately 100 µm). For each group of ten crystals, the average and standard deviation for the refined occupancy are shown separately for each of the methods used for the refinement (two conventional <i>PHENIX</i> refinements, three electron counting methods described in §2.2, and the average of these three). Since the crystals were soaked overnight (t→∞ so that O_max = O_refine) the intra-crystalline dissociation constant K_d^cryst is readily obtained from O_max using Eq. 2 (shown in the third column) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101036#pone.0101036-Danley1" target="_blank">[20]</a>. There is a significant discrepancy between the K_d^cryst value obtained from the curve fitted O_max (7.5 mM) and the value from the overnight soak O_max (17 mM). The occupancy refinement protocols all have higher precision (as seen by the low standard deviation) than accuracy. This high precision is sufficient to demonstrate that smaller crystals reach high occupancy faster. We report K_d^cryst to confirm that the binding affinity is within the expected range for a small molecule product, but with significant uncertainty. We did not perform a similar analysis for lysozyme binding to N-acetyl glucosamine because the value obtained from the curve fitted O_max (5.4 mM) was very close to reported values (4–6 mM) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101036#pone.0101036-Dahlquist1" target="_blank">[22]</a>.</p

Electron density for NAG bound to lysozyme and for ASN bound to thermolysin.

<p>Panel A: N-acetyl glucosamine is shown bound to lysozyme (difference omit map is contoured at 3.0 σ). The lysozyme data comes from a 310 µm crystal that was soaked for 750 seconds, with a refined occupancy of 74% and occupancy calculated using Eq. 1 of 68%. Panel B: Asparagine is shown bound to thermolysin (difference omit map is countered at 3.0 σ). The thermolysin data comes from a 220 µm crystal that was soaked for 601 seconds, with a refined occupancy of 99% and occupancy calculated using Eq. 1 of 84%.</p

Crystallization conditions.

<p>Crystallization conditions.</p

Refined occupancies (y axes, %) as a function of soak time (x axes, seconds).

<p>Two dimensional slices are shown for the three dimensional relationship between crystal size, ligand soak time, and occupancy (O_calc and O_refine). In each panel, the crystal size variable is excluded by grouping crystals of similar sizes. Lysozyme + NAG crystals are grouped by size (0–60 µm in box <b>A</b>, 60–120 µm in box <b>B</b>, 120–180 µm in box <b>C</b>, 180–240 µm in box <b>D</b>, 240–360 µm in box <b>E</b>, 360–480 µm in box <b>F</b>). Thermolysin + asparagine crystals are grouped into two sizes (0–150 µm in box <b>α</b>, and 150–300 µm in box <b>β</b>). Each data point represents the observed soak time and occupancy of one crystal + ligand. The average size for crystals in each range is indicated. The average number of calculated structure factors that were added into the data () is also shown (larger crystals had more overloads and consequently more added reflections). Inspection of the relationship between soak time and refined occupancy revealed a linear relationship between crystal length and the time needed to reach 50% maximum occupancy (t_1/2), so that t_1/2 = Lτ, where L is the crystal length and τ is a fixed constant. Best fits for lysozyme (R² = 78%) and thermolysin (R² = 88%) were calculated using least squares applied to Eq. 1. In each panel, a solid line shows Eq. 1 with the average size of crystals in that panel assigned to L (fitting parameters taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101036#pone-0101036-t002" target="_blank">Table 2</a>). Note that the data in each panel come from crystals with similar but not identical sizes. Consequently, the data fit Eq. 1 much better than these graphs suggest. The average residual between calculated occupancies from Eq. 1 and refined occupancies from the X-ray diffraction data was 9.76% for lysozyme + NAG and 6.51% for thermolysin + ASN.</p

Lysozyme crystals have a cubic habit and thermolysin crystals have an elongated habit.

<p>Lysozyme forms cubic crystals which were measured along the longest sides as shown (panel A). One large and one medium sized lysozyme crystal are shown. Thermolysin crystals have an elongated habit and were measured along the long axis (panel B). One large and one small crystal are shown. Occasionally a small piece of a crystal broke off (yellow highlight). In these cases, the longest crystal fragment was measured (without adjusting the length to account for the missing piece). The soaking time should correlate with the shortest crystal dimension, but the short side is difficult to measure accurately. Fortuitously, it was possible to grow lysozyme and thermolysin crystals with a very consistent crystal habit. The long crystal axis (which was easy to measure) was a good proxy way to compare the short crystal axis (which was difficult to measure).</p