Search

Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s−1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening. [ABSTRACT FROM AUTHOR]

The article presents an analysis on the new capabilities installed at National Synchroton Light Source (NSLS) beamline X26-C to support single-crystal electronic absorption and Raman spectroscopies correlated with X-ray diffraction. It says that the beamline is devoted to multidisciplinary studies having goals that include determining the relationship between the atomic and electronic structures in macromolecules. It also cites the installation of Raman spectroscopy into the beamline.

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. [ABSTRACT FROM AUTHOR]

Triplet phases recorded from insulin crystals were used to measure the improvement of phases during model refinement and to quantify the contribution made by each step in the refinement. Conventional amplitude data were recorded to 1.5 Å resolution from rhombothedral pig insulin crystals using 1.54 Å Cu Kα radiation. An initial atomic model and starting phases were obtained from a published structure and the atomic model was refined against the amplitude data using CNS. The refined phases were compared with 800 triplet phases that were measured from similar crystals using a three-beam interference technique and 1.1 Å wavelength synchrotron radiation. The solvent region was improved further using a novel density-modification procedure. Calculated triplet phases were obtained from the model after each step in the refinement and were compared with the recorded triplet phases. The average difference between the recorded triplet phases and the calculated triplet phases was used as an unbiased measure of the correctness of the model at each stage in the refinement. The average individual phase error was estimated from discrepancies from triplet phases after each refinement step. Conventional atomic refinement of an approximate starting model reduced the average individual phase error from 21.6 to 14.7°. Improvement of the solvent region, including the difference-map flattening procedure, reduced the individual phase error by a further 2.6° Modeling the discrete disorder of four amino acids accounted for an additional 0.5° improvement and the final individual phase error was 11.6°. [ABSTRACT FROM AUTHOR]

The crystal structure of the human adenovirus proteinase (AVP), a cysteine proteinase covalently bound to its 11-amino-acid peptide cofactor pVIc, has been solved to 1.6-Å resolution with a crystallographic R-factor of 0.136, Rfree=0.179. The fold of AVP–pVIc is new and the structural basis for it is described in detail. The polypeptide chain of AVP folds into two domains. One domain contains a five-strand β-sheet with two peripheral α-helices; this region represents the hydrophobic core of the protein. A second domain contains the N terminus, several C-terminal α-helices, and a small peripheral anti-parallel β-sheet. The domains interact through an extended polar interface. pVIc spans the two domains like a strap, its C-terminal portion forming a sixth strand on the β-sheet. The active site is in a long, deep groove located between the two domains. Portions are structurally similar to the active site of the prototypical cysteine proteinase papain, especially some of the Cα backbone atoms (r.m.s. deviation of 0.354 Å for 12 Cα atoms). The active-site nucleophile of AVP, the conserved Cys122, was shown to have a pKa of 4.5, close to the pKa of 3.0 for the nucleophile of papain, suggesting that a similar ion pair arrangement with His54 may be present in AVP–pVIc. The interactions between AVP and pVIc include 24 non-β-strand hydrogen bonds, six β-strand hydrogen bonds and one covalent bond. Of the 204 amino acid residues in AVP, 33 are conserved among the many serotypes of adenovirus, and these aid in forming the active site groove, are involved in substrate specificity or interact between secondary structure elements. [Copyright &y& Elsevier]