Jeffrey J. Lovelace

Tracking reflections through cryogenic cooling with topography

The mosaic structure of a single protein crystal was analyzed by reflection profiling and topography using highly parallel and monochromatic synchrotron radiation. Fine-φ-sliced diffraction images (0.002° stills) were collected using a conventional large-area CCD detector in order to calculate reflection profiles. Fine-φ-sliced topographic data (0.002°) stills were collected with a digital topography system for three reflections in a region where the Lorentz effect was minimized. At room temperature, several different mosaic domains were clearly visible within the crystal. Without altering the crystal orientation, the crystal was cryogenically frozen (cryocooled) and the experiment was repeated for the same three reflections. Topographs at cryogenic temperatures reveal a significantly increased mosaicity, while the original domain structure is maintained. A model for the observed changes during cryocooling is presented.

A very simple spiking neuron model that allows for modeling of large, complex systems

This letter introduces a biologically inspired very simple spiking neuron model. The model retains only crucial aspects of biological neurons: a network of time-delayed weighted connections to other neurons, a threshold-based generation of action potentials, action potential frequency proportional to stimulus intensity, and interneuron communication that occurs with time-varying potentials that last longer than the associated action potentials. The key difference between this model and existing spiking neuron models is its great simplicity: it is basically a collection of linear and discontinuous functions with no differential equations to solve. The models ability to operate in a complex network was tested by using it as a basis of a network implementing a hypothetical echolocation system. The system consists of an emitter and two receivers. The outputs of the receivers are connected to a network of spiking neurons (using the proposed model) to form a detection grid that acts as a map of object locations in space. The network uses differences in the arrival times of the signals to determine the azimuthal angle of the source and time of flight to calculate the distance. The activation patterns observed indicate that for a network of spiking neurons, which uses only time delays to determine source locations, the spatial discrimination varies with the number and relative spacing of objects. These results are similar to those observed in animals that use echolocation.

First results of digital topography applied to macromolecular crystals

An inexpensive digital CCD camera was used to record X-ray topographs directly from large imperfect crystals of cubic insulin. The topographs recorded were not as detailed as those which can be measured with film or emulsion plates, but do show great promise. Six reflections were recorded using a set of finely spaced stills encompassing the rocking curve of each reflection. A complete topographic reflection profile could be digitally imaged in minutes. Interesting and complex internal structure was observed by this technique. The CCD chip used in the camera has anti-blooming circuitry and produced good data quality, even when pixels became overloaded.

Advances in digital topography for characterizing imperfections in protein crystals

A system which joins digital topography with fine φ-sliced reflection profiling has been developed and applied to cryocrystallography. In this demonstration, fifteen fine φ-sliced reflection profiles with corresponding topographic sequences are evaluated: twelve reflections from a crystal at cryogenic temperatures and three reflections from a room-temperature crystal. The digitally collected data show results comparable with film, albeit at a lower resolution, but are acquired at a substantially higher rate. Additionally, anti-blooming circuitry in the CCD was tested and shown to provide useful data even when pixels were overloaded.

Physical and structural studies on the cryocooling of insulin crystals

Reflection profiles were analyzed from microgravity-grown ( micro g) and earth-grown insulin crystals to measure mosaicity (eta) and to reveal mosaic domain structure and composition. The effects of cryocooling on single-domain and multi-domain crystals were compared. The effects of cryocooling on insulin structure were also re-examined. Microgravity crystals were of larger volume, were more homogeneous and were of higher quality than earth crystals. Several micro g crystals contained a single mosaic domain which encompassed all or nearly all of the crystal with an eta(avg) of 0.005 degrees. The earth crystals varied in quality and all contained multiple domains with an eta(avg) of 0.031 degrees. Cryocooling caused a 43-fold increase in eta for micro g crystals (eta(avg) = 0.217 degrees ) and an eightfold increase for earth crystals (eta(avg) = 0.246 degrees ). These results indicate that very well ordered crystals are not completely protected from the stresses associated with cryocooling, especially when structural perturbations occur. However, there were differences in the reflection profiles. For multi-mosaic domain crystals, each domain individually broadened and separated from the other domains upon cryocooling. Cryocooling did not cause an increase in the number of domains. A crystal composed of a single domain retained this domain structure and the reflection profiles simply broadened. Therefore, an improved signal-to-noise ratio for each reflection was measured from cryocooled single-domain crystals relative to cryocooled multi-domain crystals. The improved signal from micro g crystals, along with the increase in crystal size, facilitated the measurement of the weaker high-resolution reflections. The observed broadening of reflection profiles indicates increased variation in unit-cell parameters, which may be linked to cryocooling-associated structural changes and disorder.

Imaging modulated reflections from a semi-crystalline state of profilin:actin crystals

Modulated protein crystals remain terra incognita for most crystallographers. While small-molecule crystallographers have successfully wrestled with and conquered this type of structure determination, to date no modulated macromolecular structures have been reported. Profilin:β-actin in a modulated semi-crystalline state presents a challenge of sufficient biological significance to motivate the development of methods for the accurate collection of data on the complex diffraction pattern and, ultimately, the solution of its structure. In the present work, fine φ-sliced data collection was used to resolve the closely spaced satellite reflections from these polymorphic crystals. Image-processing methods were used to visualize these data for comparison with the original precession data. These preliminary data demonstrate the feasibility of using fine φ-slicing to collect accurately the intensities and positions of the main and satellite reflections from these modulated protein crystals.

The use of orthogonal projection to visualize mosaic domains from topographic data collected on protein crystals

Owing to the relatively low intensity of diffraction and resulting low contrast topographic images, and also the richly detailed domain structure, it can be difficult to extract individual mosaic domains (size and shape) for protein crystals. Here, orthogonal projection was tested and found to be a superior approach over previous methods to extract mosaic domain information from a fine-sliced topographic sequence of images. The topographic sequence was collected at room temperature from a lysozyme crystal with a low mosaicity. Orthogonal projection can be applied to image sequences that are spatially invariant and composed of linearly additive image formation processes. The domains were determined by using a particle swarm optimizer to fit Gaussians to the integrated intensity profile of the reflection as a function of angle, although any basis function could have been used. This optimization method was more amenable to automation and converged to the global minimum more efficiently. The number of domains can normally be determined by visually inspecting the integrated intensity profile; this only needs to be done once for each crystal, because the number of domains remains constant during collection, and not for each reflection. The results can be used to provide a picture of the internal structure of the crystal and may be useful to aid in the improvement of crystal growth techniques.

Superspace collision: a higher-dimensional framework describes unexpected supercell refinement results

Protein crystals can be modulated1. Modulations come in two varieties: commensurate and incommensurate. Data with a commensurate modulation can be indexed and integrated using a supercell and refined using standard approaches. Incommensurate data must be handled using superspace methods2. In superspace, for the 4D case, atoms are described as lines that follow a periodic path described by an atomic modulation function (AMF). The superspace approach is also valid for a commensurate modulation but the supercell pathway is much easier and currently the only option available leaving incommensurate data unsolvable for proteins at this time.