1. Lecture 53: X-ray crystallography

2. Electrons deflect x-rays We try to recreate electron density from the x-ray diffraction pattern Each point in space has an average electron density – Need 4Ds to plot it in full! – Instead, plot all points with the same density: forms a surface

3. What resolution do we need to model our protein’s structure? Given an electron density, we would like to be able to identify side chains so we can locate each residue of the protein. Typical covalent bond length: 1 Angstrom At 1.2 Angstrom resolution, we can make out individual atoms. At up to 3 angstroms, we can see at least large side chains.

4. What is a crystal?

5. Why do we need a crystal? Signal enhancement – Scattering from one protein would be too weak to detect Immobilization – Don’t allow atoms to move while we are attempting to determine their position! Alignment – Aligned proteins will all diffract incident x-rays in the same way vs.

6. How is the crystal produced? No magic formula, but some options include: Adding precipitants (additives that increase the effective [protein] by coordinating solvent) Providing a seed crystal Tuning the pH to minimize a protein’s net charge (decreases protein-protein repulsion) Study fragments (remove disordered regions)

7. In practice, we test a wide variety of conditions in very small volumes Mosquito liquid handler

8. What kind of crystal do we obtain when working with proteins? On average, 50% solvent – Fills in gaps between densely-packed proteins – Some solvent molecules may be immobile (but most are not) 2-500 microns thick for x-ray diffraction – (If electron diffraction, can get away with 0.5-1  m)

9. Does crystallization affect a protein’s folded structure? Usually not, but worth checking: Enzymes are often still active in the crystal – Substrate and product can flow in/out via solvent gaps Site-directed mutagenesis can be used to confirm predictions from crystal structures Crystal structures often reflect predictions from NMR and other native structure determination techniques

10. Where do we get X-rays? Advanced Photon Source at Argonne National Labs Synchrotrons Rotating metal anode

11. How do we perform X-ray diffraction with our crystal?

13. A typical diffraction pattern

14. According to Bragg’s Law, we have full constructive interference when: Will the minimum angle  be biggest when a is large, or when a is small? Where are these diffraction spots coming from?

15. A typical diffraction pattern Diffraction spots from atoms which are close together are out at the edge of the detector: we need this data to get a high resolution! Diffraction spots have different intensities: Why? Maximum angle determines the resolution:

16. Can we use this understanding to interpret Photo 51 (B-form DNA)? Incident x-rays Crystal orientation

17. Can we use this understanding to interpret Photo 51 (B-form DNA)? Most distant: spacing between adjacent nucleotides/phosphates Incident x-rays Crystal orientation Less distant: larger spacings between bases  

18. Can we use this understanding to interpret Photo 51 (B-form DNA)? Line missing due to interference from second helical repeat Incident x-rays Crystal orientation

19. Can we use this understanding to interpret Photo 51 (B-form DNA)? a b c d a = 3.4 Å c = 20 Å d = 20 Å b = 34 Å