J.G. Grossmann

Structural changes in α-crystallin and whole eye lens during heating, observed by low-angle X-ray diffraction

Abstract Whole eye lens and α-crystallin gels and solutions were investigated using X-ray scattering techniques at temperatures ranging from 20 °C to 70 °C. In whole lens isolated in phosphate-buffered saline, the spacing of the dominant X-ray reflection seen with low-angle scattering was constant from 20 °C to 45 °C but increased at 50 °C from 15.2 nm to 16.5 nm. At room temperature, the small-angle X-ray diffraction pattern of the intact lens was very similar to the pattern of α-crystallin gels at near-physiological concentration (≈300 mg/ml), so it is reasonable to assume that the α-crystallin pattern dominates the pattern of the intact lens. Our results therefore indicate that in whole lens α-crystallin is capable of maintaining its structural properties over a wide range of temperature. This property would be useful in providing protection for other lens proteins super-aggregating. In the α-crystallin gels, a moderate increase in both the spacing and intensity of the reflection was observed from 20 °C to 45 °C, followed by an accelerated increase from 45 °C to 70 °C. Upon cooling, this effect was found to be irreversible over 11 hours. Qualitatively similar results were observed for α-crystallin solutions at a variety of lower concentrations.

Ab initio phasing using molecular envelope from solution X-ray scattering.

Solving the phase problem is the crucial and quite often the most difficult and time-consuming step in crystallographic structure determination. The traditional methods of isomorphous replacement (MIR or SIR) and molecular replacement require the availability of an isomorphous heavy-atom derivative or the structure of a homologous protein, respectively. Here, a method is presented which utilizes the low-resolution molecular shape determined from solution X-ray scattering data for the molecular search. The molecular shape of a protein is an important structural property and can be determined directly by the small-angle scattering technique. The idea of locating this molecular shape in the crystallographic unit cell has been tested with experimental diffraction data from nitrite reductase (NiR). The conventional Patterson search proved to be unsuccessful, as the intra-envelope vectors are uniformly distributed and do not match those of intra-molecular (atom-to-atom) vectors. A direct real-space search for orientation and translation was then performed. A self-rotation function using 2.8 A crystallographic data yielded the polar angles of the non-crystallographic threefold axis. Knowledge of the orientation of this axis reduces the potential six-dimensional search to four (Eulerian angle gamma and three translational parameters). The direct four-dimensional search within the unit cell produced a clear solution. The electron-density map based on this solution agrees well with the known structure, and the phase error calculated from the map was 61 degrees within 20 A resolution. It is anticipated that the low-resolution envelope can be used as a starting model for phase extension by the maximum-entropy and density-modification method.

Can hair be used to screen for breast cancer

Editor—The use of hair as a biopsy tissue has been considered for some time. For instance, in the case of breast cancer, raised zinc levels in head hair have been reported.1 Besides, x ray diffraction patterns of hair are rich and have attracted much attention for 70 years.2 However, its potential use as a diagnostic indicator of disease was only suggested a short time ago.3 Most recently, James et al 4 reported that x ray diffraction of hair taken from women diagnosed with breast cancer (and those at high risk by virtue of a proven BRCA1 / BRCA2 mutation) showed a diffuse ring. They claimed a 100% correlation with the disease, advocating the use of pubic hair as a simple non-invasive screening method for breast cancer. The use of pubic hair was suggested in view of possible damage to the head hair from cosmetic treatments. Despite this note of caution, the study of James et al 4 was based on 12 pubic hair samples with only eight from cancer affected subjects. Here, we report a detailed double blind study from 109 women belonging to five clinically distinct groups as well …

Complementing structural information of modular proteins with small angle neutron scattering and contrast variation

AbstractMany macromolecules in the cell function by forming multi-component assemblies. We have applied the technique of small angle neutron scattering to study a nucleic acid–protein complex and a multi-protein complex. The results illustrate the versatility and applicability of the method to study macromolecular assemblies. The neutron scattering experiments, complementing X-ray solution scattering data, reveal that the conserved catalytic domain of RNase E, an essential ribonuclease in Escherichia coli (E. coli), undergoes a marked conformational change upon binding a 5′monophosphate–RNA substrate analogue. This provides the first evidence in support of an allosteric mechanism that brings about RNA substrate cleavage. Neutron contrast variation of the multi-protein TIM10 complex, a mitochondrial chaperone assembly comprising the subunits Tim9 and Tim10, has been used to determine a low-resolution shape reconstruction of the complex, highlighting the integral subunit organization. It shows characteristic features involving protrusions that could be assigned to the six subunits forming the complex.

Implementation of cluster analysis for ab initio phasing using the molecular envelope from solution X-ray scattering

Solution of the phase problem is central to crystallographic structure determination. The conventional methods of isomorphous replacement (MIR or SIR) and molecular replacement are ineffective in the absence of a suitable isomorphous heavy-atom derivative or knowledge of the structure of a homologous protein. A recent method utilizing the low-resolution molecular shape determined from solution X-ray scattering data has shown to be successful in locating the molecular shape within the crystallographic unit cell in the case of the trimer nitrite reductase (NiR, 105 kDa) [Hao et al. (1999), Acta Cryst. D55, 243-246]. This was achieved by performing a direct real-space search for orientation and translation using knowledge of the orientation of the polar angles of the non-crystallographic axis obtained by performing a self-rotation on crystallographic data. This effectively reduces the potential six-dimensional search to a four-dimensional one (Eulerian angle gamma and three translational parameters). In the case of NiR, the direct four-dimensional search produced a clear solution that was in good agreement with the known structure. The program FSEARCH incorporating this method has been generalized to handle molecules from all space groups and in particular those in possession of non-crystallographic symmetry. However, the method employed was initially unsuccessful when applied to the small dimeric molecule superoxide dismutase (SOD, 32 kDa) owing to the absence of strong reflections at low resolution caused by saturation at the detector. The determined solution deviated greatly from that of the known structure [Hough & Hasnain (1999), J. Mol. Biol. 287, 579-592]. It was found that once these absent reflections were replaced by a series of randomly generated intensity values and cluster analysis was performed on the output, the signal-to-noise ratio was improved and a most probable solution was found. The electron-density map of the stochastically determined solution agrees well with the known structure; the phase error calculated from this map was 67 degrees within 14 A resolution.

3-Carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa: MAD phasing with 80 selenomethionines

The structure of 3-carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa was determined at 3.0 A resolution. Phase information was derived from a multiwavelength anomalous dispersion (MAD) experiment conducted at three wavelengths using crystals of fully substituted selenomethionine protein. However, the structure determination was not routine owing to the relatively poor quality of the diffraction data and the large number of twofolds in the unit cell. Eventually, 80 selenium sites were identified by the combined use of direct methods and real-space map interpretation. This represents one of the largest selenium substructures solved and used for phasing. Some of the difficulties in the structure determination and the methods used to address them are discussed.

Structure of Klebsiella pneumoniae Nitrogenase

The 3D crystallographic structure of the nitrogenase MoFe protein was first determined for the protein from Azotobacter vinelandii (Av1) at 2.8 A resolution and identified two unique metal-sulphur clusters viz the FeMoco centres and the P clusters (Kim, Rees 1992). This structure was later refined to 2.2 A resolution (Chan et al, 1993). Analysis of the structure of the MoFe protein from Clostridium pasteurianum (Cpl) was consistent with the formulation of FeMoco as MoFe7S9. homocitrate but differed in the interpretation of the structure of the P clusters (Bolin et al, 1993). Rees’s group reported that the P clusters consisted of two Fe4S4 clusters bonded through a disulphide bridge at one corner whereas Bolin suggested that the two Fe4S4 cubanes shared a single sulphur atom at the corner to produce an Fe8S7 cluster. (Fig 1)

Molecular Conformations of Nitrogenase Enzymes and Their Complexes in Solution

Formation of a complex between the component proteins of nitrogenase is an essential step in the catalytic cycle. X-ray scattering has been used to probe the structure of a transition state complex of the proteins from Klebsiella pneumoniae (Kp) as Kp1-Kp2-MgADP-AlF4 (Grossmann et al., 1997) and of the individual proteins in aqueous solution.