Dataset Information

Acoustic methods for high-throughput protein crystal mounting at next-generation macromolecular crystallographic beamlines.

ABSTRACT: To take full advantage of advanced data collection techniques and high beam flux at next-generation macromolecular crystallography beamlines, rapid and reliable methods will be needed to mount and align many samples per second. One approach is to use an acoustic ejector to eject crystal-containing droplets onto a solid X-ray transparent surface, which can then be positioned and rotated for data collection. Proof-of-concept experiments were conducted at the National Synchrotron Light Source on thermolysin crystals acoustically ejected onto a polyimide `conveyor belt'. Small wedges of data were collected on each crystal, and a complete dataset was assembled from a well diffracting subset of these crystals. Future developments and implementation will focus on achieving ejection and translation of single droplets at a rate of over one hundred per second.

SUBMITTER: Roessler CG

PROVIDER: S-EPMC3747951 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Publications

Acoustic methods for high-throughput protein crystal mounting at next-generation macromolecular crystallographic beamlines.

Roessler Christian G CG Kuczewski Anthony A Stearns Richard R Ellson Richard R Olechno Joseph J Orville Allen M AM Allaire Marc M Soares Alexei S AS Héroux Annie A

Journal of synchrotron radiation 20130808 Pt 5

To take full advantage of advanced data collection techniques and high beam flux at next-generation macromolecular crystallography beamlines, rapid and reliable methods will be needed to mount and align many samples per second. One approach is to use an acoustic ejector to eject crystal-containing droplets onto a solid X-ray transparent surface, which can then be positioned and rotated for data collection. Proof-of-concept experiments were conducted at the National Synchrotron Light Source on th ...[more]

PMID: 23955046

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Project description:Conventional macromolecular crystallographic refinement relies on often dubious stereochemical restraints, the preparation of which often requires human validation for unusual species, and on rudimentary energy functionals that are devoid of nonbonding effects owing to electrostatics, polarization, charge transfer or even hydrogen bonding. While this approach has served the crystallographic community for decades, as structure-based drug design/discovery (SBDD) has grown in prominence it has become clear that these conventional methods are less rigorous than they need to be in order to produce properly predictive protein-ligand models, and that the human intervention that is required to successfully treat ligands and other unusual chemistries found in SBDD often precludes high-throughput, automated refinement. Recently, plugins to the Python-based Hierarchical ENvironment for Integrated Xtallography (PHENIX) crystallographic platform have been developed to augment conventional methods with the in situ use of quantum mechanics (QM) applied to ligand(s) along with the surrounding active site(s) at each step of refinement [Borbulevych et al. (2014), Acta Cryst D70, 1233-1247]. This method (Region-QM) significantly increases the accuracy of the X-ray refinement process, and this approach is now used, coupled with experimental density, to accurately determine protonation states, binding modes, ring-flip states, water positions and so on. In the present work, this approach is expanded to include a more rigorous treatment of the entire structure, including the ligand(s), the associated active site(s) and the entire protein, using a fully automated, mixed quantum-mechanics/molecular-mechanics (QM/MM) Hamiltonian recently implemented in the DivCon package. This approach was validated through the automatic treatment of a population of 80 protein-ligand structures chosen from the Astex Diverse Set. Across the entire population, this method results in an average 3.5-fold reduction in ligand strain and a 4.5-fold improvement in MolProbity clashscore, as well as improvements in Ramachandran and rotamer outlier analyses. Overall, these results demonstrate that the use of a structure-wide QM/MM Hamiltonian exhibits improvements in the local structural chemistry of the ligand similar to Region-QM refinement but with significant improvements in the overall structure beyond the active site.

Dataset Information

Acoustic methods for high-throughput protein crystal mounting at next-generation macromolecular crystallographic beamlines.

Publications

Acoustic methods for high-throughput protein crystal mounting at next-generation macromolecular crystallographic beamlines.

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