Improving the diffraction of full-length human selenomethionyl metavinculin crystals by streak-seeding

Abstract

Metavinculin is an alternatively spliced isoform of vinculin that has a 68-residue insert in its tail domain (1134 total residues) and is exclusively expressed in cardiac and smooth muscle tissue, where it plays important roles in myocyte adhesion complexes. Mutations in the metavinculin-specific insert are associated with dilated cardiomyopathy (DCM) in man. Crystals of a DCM-associated mutant of full-length selenomethionine-labeled metavinculin grown by hanging-drop vapor diffusion diffracted poorly and were highly sensitive to radiation, preventing the collection of a complete X-ray diffraction data set at the highest possible resolution. Streak-seeding markedly improved the stability, crystal-growth rate and diffraction quality of DCM-associated mutant metavinculin crystals, allowing complete data collection to 3.9 Å resolution. These crystals belonged to space group P4(3)2(1)2, with two molecules in the asymmetric unit and unit-cell parameters a=b=170, c=211 Å, α=β=γ=90°.

Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin

“…Vinculin was also shown to regulate the mechanical stress of the extracellular environment and to function as a mechano‐coupling protein 9. Our human full‐length 117 kDa vinculin and 124 kDa metavinculin crystal structures10–12 showed that the globular vinculin head (VH) domain is comprised of three seven‐helix bundles (Vh1–Vh3) and a four‐helix bundle (Vt2). A proline‐rich hinge region links VH and to a five‐helix bundle tail domain.…”

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

“…By transferring nucleated material from one condition to another, one may attempt to effectively decouple these processes, thus giving access to new, as yet unexplored crystallization space, and as a result increasing the overall success rate of a screening experiment. Established methods have been documented for (i) macroseeding, the transfer of a single crystal in its entirety from one condition to another 8 , (ii) streak seeding, the transfer of nucleated material, generally obtained by the application of directional pressure using for example a cat's whisker to the surface of an existing crystal, followed by subsequent passage of the whisker through a new crystallization drop 9 , and (iii) "classical" microseeding, the transfer of crystal "seeds", generated by harvesting crushed crystals (or crystalline material), into conditions similar to those that yielded the seeds 10 . Notably all three of these methods are time consuming and poorly scalable, certainly in comparison to what is achievable with modern liquid handling crystallization robotics.…”

Crystallization and preliminary X-ray studies of ferric uptake regulator fromMagnetospirillum gryphiswaldense

“…However, these crystals were small and were unsuitable for diffraction studies; for example, the resolution of the rod-shaped crystals was about 10 Å . To improve the crystal quality, the seeding technique (Rangarajan & Izard, 2010) was used. A seed stock was made by crushing two rod-shaped crystals in the mother liquor and was diluted 1000-10 000 times.…”