1. Ultra-High Resolution X-ray Diffraction
12 X-ray diffraction data sets collected at the APS
pH 5
pH 7
pH 9
Room temperature, hydrogenated
1.05 Å, R=10.6%
1.05 Å, R=11.3%
1.15 Å, R=11.1%
Room temperature, deuterated
1.05 Å, R=10.7%
1.05 Å, R=12.2%
1.20 Å, R=10.4%
Liquid nitrogen temperature, hydrogenated
1.05 Å, R=10.9%
1.05 Å, R=11.5%
1.05 Å, R=11.4%
Liquid nitrogen temperature, deuterated
1.10 Å, R=10.6%
1.00 Å, R=10.9%
1.05 Å, R=11.9%
Mention that these are refined using totally unrestrained refinement, so there is no geometric bias as to how the atoms should behave, it is dictated totally by the electron density

2. Ultra-High Resolution X-ray Diffraction
Covalent distances
Measure C—O and C—N lengths for ionizable amino acids
The Cambridge Structural Database
Small molecule structures; 1.00 Å resolution or better
Asp/Glu Example
Non-covalent distances
Hydrogen bonding
C double bond O distance and C single bond OH distance are 1.20 and 1.30 A, respectively. When electrons are de-localized, see C/O distances of 1.25 A for each C-O bond. With the precision of the data that we have, we should be able to tell the difference.
1.20 Å
1.25 Å
1.30 Å
1.25 Å

3. Oops
Recent NMR work on alpha-lytic protease established the pKa of the active site Asp in the serine proteases as being around 2. Should therefore see no proton on this residue at pH 5. In our room temperature structures of gamma-chymotrypsin, it is indeed unprotonated.
BUT – in the cryo structures, it is protonated at pH 5.

4. Precision and Accuracy of the Distance Data
Calculate RMSD of all bonds for individual residues
At highest resolution (1.00 Å), differences greater than 0.02 Å are significant
BUT – need “gold standard” where we are CERTAIN of the protonation state of all ionizable residues
SOLUTION – NEUTRON DIFFRACTION to locate deuterons directly from their nuclear density, at room temperature, at pH 5, 7 and 9
For individual residues, as those values will more closely represent deviations from the distances that we will be measuring here

5. Asparagines are Unambiguous
Nuclear Density
Electron Density

6. Water Orientation
Nuclear Density = Blue Electron Density = Red

7. Structure 22, 899–910, June 10, 2014

9. To Summarize
Nearly all the structures in the Protein Data Bank in the last years have been determined at -200oC, more than 100 degrees below the glass transition in protein dynamics
Such low temperatures have clear-cut effects on protein dynamics and protein volume and internal packing, but indeterminate effects on loop and side-chain conformations, inter-domain positions, residue protonation states, and metal ion spin states, among other things

10. A Proposal
The protein crystallographic community should make a concerted effort to determine at least one room temperature crystal structure for every unique protein in the Protein Data Bank, at the highest possible resolution

11. A Proposal
The protein crystallographic community should make a concerted effort to determine at least one room temperature crystal structure for every unique protein in the Protein Data Bank, at the highest possible resolution. This might be a very good use for Free Electron Laser crystallography

15. Tom Alber