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Strategies for expression and solubility analysis

Production of soluble protein is one of the major bottlenecks that precede crystallographic studies.During the last years several techniques and strategies have been developed to address this problem. However, many of them imply an economical cost and technologies that are not always available. We will describe a general plan for protein solubility analysis by using a combination of four different but complementary strategies. In this plan, different constructs of a protein interest are designed

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Automating Microseeding Protein Crystallography Set-ups Using Mosquito
Joby Jenkins, David Smith, Rob Lewis, Chloe Carter

Crystallising proteins, required for structure determination by X-ray diffraction, is a difficult and labour-intensive task. The mosquito liquid handler (TTP LabTech) is ideally suited to automating the complex set-ups required for microseeding due to its precide handling of extremely low volumes of even viscous solutions, and its ability to perform multiple aspirations and dispenses with each pipette.

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Automated Single Crystal Structure Determination - A Tool for Synthetic Chemists?
Bernd Hinrichsen, Martin Adam, Michael Carr, Dieter Schollmeyer,

During recent years large improvements in software functionality and its ease-of-use have made single crystal X-ray structure determination easier than ever. These days most structures can be measured, processed, solved and refined using well selected defaults with no or little crystallographic knowledge. Recently, microfocus sources and CCD detectors both air-cooled, have entered the marketplace. Combining these innovations with an automated sample loader and an intelligent graphical user inte

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Advances in Crystallographic Hardware for Structural Biology
Matthew Benning and Michael Ruf, Bruker AXS Inc., East Cheryl Parkway

The number of life science investigators utilizing crystallography in their research increases every year. Synchrotrons are an invaluable resource for structural work but not everyone has unlimited access to a beamline. In-house diffraction systems complement synchrotron access and can provide increased productivity in the home lab. In recent years, the performance and versatility of in-house systems has greatly improved. The enhanced performance allows data collection on demanding projects such

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MED-SuMo and MED-Hybridise: exploit all PDB macromolecule structures to design/optimize innovative leads
Moriaud F., Oguievetskaia K., Adcock S.A., Vorotynsev A.M., Martin L., Doppelt-Azeroual O. and Delfaud F.

Obtaining structural information on fragment-protein target is a key factor and also a major limitation. We’ve used MED-SuMo and MED-Hybridise to query and mine the PDB seeking for similar interaction surfaces. MED-portions were used to design novel scaffolds (GPCR) and decorate a given scaffold (kinase). We have analysed the scaffolds in regard to their diversity, their presence in PubChem, PDB and other libraries.

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Phase Diagram Visualization via Continuously-Fed Crystallization: Experiments and Model
Masano Sugiyama and Victor Barocas

A continuously-fed crystallization chamber is manufactured to allow phase diagram visualization. This microfluidic system allows the experimenter to screen a large range of salt and protein concentrations in one experiment. This allows the experimenter to predict the protein phase diagram in a single experiment. A continuous-feed crystallization chamber has been successfully fabricated and characterized in terms of its flow profile, and has successfully predicted the phase diagram for lysozyme.

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Low Volume Liquid Handling of Organic Solvents for Compound Storage and Dissolution using mosquito
Gillian Lewis, Tristan Cope, Joby Jenkins, David Gledhill & Rob Lewis

mosquito® is a positive displacement liquid handling system capable of pipetting solutions with a high degree of accuracy (to within 5%) and precision (CVs of <10%) in the nanolitre range. Here, we demonstrate mosquito’s ability to dispense accurately low volumes of organic solvents of low surface tension and to re-dissolve compounds which have been previously dried in storage wells.

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Inferring synaptotagmin function from C2 domain stability: Analysis of structurally defined mutations in synaptotagmin 1 C2A
K.L Fuson, Kristofer Knutson, Anil Bhatta, Greg Gillispie, Candace Lange, Anne Hinderliter, R. Bryan Sutton

Synaptotagmin 1 (Syt1) triggers the release of neurotransmitter from docked synaptic vesicles through its interactions with Ca+2, phospholipid and the SNARE complex. We tested whether the disruption of this single H-bond can affect the shape of the Ca+2 binding pocket of C2A by causing loop 3 to collapse.

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Lead Optimization Computational Protocol at PDB Scale to Rationally Optimize Attachments to a Given Kinase Inhibitor Scaffold
Moriaud F., Henry T., Adcock S.A., Vorotynsev A.M., Martin L., Doppelt O.

We’ve used MED-SuMo to query and mine the Protein’s Surface Chemical Functions surrounding fragments of PDB ligands, seeking similarities with the kinase of interest (Vegfr DFGout, pdb code 2oh4, ligand code GIG) and collecting a library of 1129 unique fragments positioned in the vegfr’s active site and annotated with the counts of contacts and h-bonds. With them we optimize a substructure of the GIG ligand to find others DFGout ligands.

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Scientific News
Protein Crystalline Frameworks: A New Class of Materials
Scientists at the Helmholtz Center Berlin (HZB) along with researchers at China’s Fudan University have characterized a new class of materials called protein crystalline frameworks (PCFs).
Worm Virus Details Come to Light
Rice University scientists have won a race to find the crystal structure of rare nematode virus, known to infect the most abundant animal on Earth.
Structure of Molecular Machine for Targeted Viral DNA Destruction Determined
The MSU-led research provides the first detailed blueprint of a multi-subunit “molecular machinery” that bacteria use to detect and destroy invading viruses.
Flu’s Mechanisms Clues Uncovered
Researchers from Rice University and Baylor College of Medicine have analyzed how influenza-related proteins help infect cells.
Innovative Technology Enables First Crystal Structure of mGlu5 Transmembrane Domain to be Determined
Several novel drug candidates have been identified to help treat specific neuropsychiatric disorders.
Neutron Crystallography Resolves Long-Standing Molecular Mystery
Research has implications for understanding of oxidative processes within living cells which is crucially important for drug development.
Considering the ‘Anticrystal’
Researchers have evidence of a new concept, the anticrystal, a theoretical solid that is completely disordered.
How Developing Neurons Sense a Chemical Cue
An embryo organizes itself into left and right halves as it grows. But a certain set of nerve cells do something unusual: they cross from one side to the other.
High-speed Snapshots of Biomolecules
PETRA III pioneers protein serial crystallography at synchrotrons.
Stanford Researchers Discover Immune System’s Rules of Engagement
Study finds surprising similarities in the way immune system defenders bind to disease-causing invaders.
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