Strategies for Protein Cryocrystallography

Abstract

Cryoprotection is the final step before flash-cooling, during which crystals can be improved or damaged and data quality maximized. A well thoughtout cryoprotection requires optimized composition of suitable components and an appropriate soak period. Crystallization methodology has been extensively studied, but not cryoprotection. Cryoprotectant selection remains a trial and error exercise where the first combination that "works" is accepted. The approach presented here consists in a formulation of a few mixed compounds to speed-up crystal preparation for synchrotron data collection. A good cryoprotectant solution needs to stabilize the crystalline state and prevent ice formation during flash-cooling, its composition can differ widely from the crystallization precipitant since it is not required to induce nucleation, establish an equilibrium between the protein crystal and the solution or permit crystal growth. The strategy presented here differs from the general practice that involves a single anti-icing chemical, often glycerol, a molecule able to enhance protein solubility. The multicomponent mixtures selected through an evolutive process neither increase nor decrease protein solubility but provide extended stabilization during cryoprotection to allow longer soak periods without crystal cracking or dissolving.

NMR of sedimented, fibrillized, silica-entrapped and microcrystalline (metallo)proteins

“…If the use of lower temperature is required, it is also important to inhibit the formation of ice that can distort the crystal lattice up to the point of breaking long-range order [56][57][58].…”

N-O-Isopropyl Sulfonamido-Based Hydroxamates as Matrix Metalloproteinase Inhibitors: Hit Selection and in Vivo Antiangiogenic Activity

“…Prior to data collection at the Soleil synchrotron (St. Aubin, France) on beamline Proxima 1, crystals were transferred to an appropriate mixed cryoprotectant solution. 40 Data was reduced using XDS 41 and the script "xdsme". The crystal of the complex MMP-9•3 belonged to the space group P2 1 and diffracted to 1.65 Å resolution ( A table reporting the combustion analysis data of the final products, a table with statistics for MMP-9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 …”

X-ray micro-diffraction studies on biological samples at the BioCAT Beamline 18-ID at the Advanced Photon Source

“…Samples plunged in liquefied propane are pre-mounted on pins that fit into a customized Hampton haemostat where the inner surfaces are widened to accept larger sample holders than the mounts typically used for macromolecular crystallography, and transferred to the sample position in the beam in the cryo-jet using the normal cryocrystallgraphy transfer technique with a haemostat. Both flashfreezing and plunge-freezing techniques have been explained elsewhere (Vera & Stura, 2014;Warkentin et al, 2006), but for our larger fibrous samples flash-freezing requires particular care and speed in handling to prevent warming of the sample during transfer. Using either flash-freezing or plunge-freezing, we have been successful in preserving connective tissue samples in the X-ray beam for extended periods of time, up to 60 min whilst frozen, and without any obvious signs of degradation (as determined from comparison of diffraction patterns from the start, middle and end of data collection, changes in intensity and intensity peak positions being clear indicators of damage).…”