Ab initio crystallographic structure determination of insulin from protein to electron density without crystal handling

The crystallization, cryoprotection and heavy-atom derivatization of insulin have been optimized using the counter-diffusion technique in a restricted geometry. The protein crystals obtained were immediately used for in situ X-ray diffraction, phase determination by SAS and electron-density map calculation without crystal manipulation.

Granada Crystallisation Box: a new device for protein crystallisation by counter-diffusion techniques

Granada Crystallisation Box (GCB) is a new crystallisation device designed to perform counter-diffusion experiments. Here we describe the device and its use for protein crystallisation purposes. GCB allows one to explore and exploit the coupling between crystallisation and diffusion. The role of viscous fluids, gels and/or microgravity can be enhanced by using capillary volumes, creating a perfect diffusive mass transport scenario. The use of capillaries also reduces the consumption of macromolecules and avoids the handling of crystals for X-ray diffraction data collection.

Agarose as crystallisation media for proteins II: Trapping of gel fibres into the crystals

The crystallisation pressure exerted by protein crystals growing in agarose gel does not disrupt the gel network. However, protein crystals trap agarose fibres when they grow in agarose gels. The fibres of agarose are distributed randomly in the crystals explaining why they do not appreciably affect the diffraction quality of the crystal