HPC applications

Cryocooling at atmospheric pressure generally causes some degradation in diffraction quality. HPC, applied with 200-400 MPa (typically 200 MPa, or 2 kbar) of helium gas, can reduce this degradation.

HPC is worth trying if you have crystals that diffract well at room temperature, but have problems when cryocooled by normal means. These issues include degraded diffraction quality of crystals cryocooled with added cryoprotectant and the inability of finding a suitable cryoprotectant after exhaustive search.

For example, crystals of the bacterial pilin protein SpaD showed much improved diffraction when cryocooled under pressure. HPC was essential in obtaining the structure of full-length SpaD from Corynebacterium diphtheriae. This full-length structure was essential for understanding the role of isopeptide bonds in the stability and interconnection of protein modules for the assembly of the pili of Gram-positive bacteria (Kang et al. 2014, Acta Cryst D70, 1190-1201).

Improved resolution due to HPC is primarily due to two factors: (1) pressurization converts internal water in the crystal to a high density amorphous (HDA) form, which causes less disruption to the crystal lattice on cooling than the room pressure low density amorphous (LDA) form; and (2) the ability to reduce or eliminate cryoprotectant, and hence any effect that the extra solvent component could have on crystal packing.

Not all crystals show better diffraction using HPC. In particular, those that diffract extremely poorly are unlikely to be much improved; the molecules in such crystals are poorly packed in the first place. When improvement in resolution is observed, it is typically on the order of 0.5-1.0 Å.

In some cases, application of pressure can improve the X-ray diffraction quality of protein crystals. This is presumably a result of pressure favoring a more ordered packing and reduced lattice disorder of the molecules in the crystal.

The use of HPC to reduce lattice disorder has been described in (Huang et al., 2016, J. Appl. Cryst., 49, 149-157). Examples of general reduction in disorder include:

• In the endosomal sorting protein snf7, resolution improved from 2.4 to 1.6 Å. This was accompanied by a dramatic reduction in unit cell dimensions due to a shape change of the protein molecules that resulted in tighter packing (an unusual effect, as unit cell changes are generally more subtle). (Tang et al., eLife 2015 Dec 15, e 12548).



• In lpg1496, an effector protein from the Gram-negative bacterium Legionella pneumophila, HPC at 350 MPa resulted in improved resolution (~2.4 to 1.45 Å) and a large decrease in mosaic spread.



• In the O-methyltransferase Calo6, the resolution improved from 3.4 to 2.3 Å and the anisotropy of the diffraction was much reduced.



• In human glutaminase C, HPC was able to resolve non-merohedral twinning, reducing multiple twin domains to a single lattice.

Gaseous reactants can be trapped in the active site of an enzyme by pressurizing a crystal of the enzyme with the gas of interest and cryocooling. If interested, please contact Qingqiu Huang.

Our pressure cryo-cooling system can safely handle biological gases, such as carbon dioxide and oxygen, and hazardous gases, such as nitric oxide. Crystals are typically infused with a gas at 5 - 20 MPa and then immediately flash frozen. 

Gas trapping by HPC permits researchers to capture the bound state of an otherwise transient species. For example, carbonic anhydrase (CA) regulates the pH of the blood by converting carbon dioxide to bicarbonate, though the exact mechanism for this reaction had not yet been proven. Pressurizing human CA II with CO₂ using HPC helped determine the CO₂ binding site and confirm the model (1). Later work compared the CO₂ site between bacterial and human CAs (2).

References:
(1) Domsic et al. 2008, J. Biol. Chem. 45, 30766-30771
(2) Aggarwal et al. 2015, Biochemistry 54, 6631-6638

HPC causes other changes in crystals that can be leveraged for studying the interior structure of proteins.

• Pressure can affect other properties through subtle effects on protein structure. For example, the protein citrine exhibits a fluorescence peak shift from yellow to green under pressure. HPC revealed corresponding molecular changes of the chromophore under pressure (Barstow et al. 2009, Biophy. J., 97, 1719-1727).

• Pressure can also act to stabilize bound ligands, such as ATP-γS in the KtrAB K+ transporter (Albright et al. 2006, Cell 126, 1147-1159).


• Pressurizing with an inert gas, such as Kr or Xe, and then switching to He, all at ~200 Mpa, can introduce Kr or Xe in a few well-ordered sites, for phasing using the anomalous diffraction of the noble gas atoms (Kim et al. 2006, Acta Cryst. D62, 687-694).

• Pressure affects not only the macromolecules in a crystal, but also water in the lattice. Experiments using HPC have increased our knowledge of high-density to low-density amorphous ice transitions (Kim et al. 2009, PNAS 106, 4596-4600).