1. Considerations for Protein Crystallography (BT Chapter 18) 1.Growing crystals Usually require 0.5mm in shortest dimension, except if using Synchrotron radiation; Can be “twinned” (two or more crystals growing into each other) Why X-rays? The wavelength of radiation is comparable with the length of interatomic distances.

2. Considerations for Protein Crystallography 2.Collecting Diffraction Data Synchrotron radiation has become routine for structural determinations. The high intensity beam reduces both the time to collect data and size of crystal needed.

3. Defining the unit cell of the crystal Figures 18.6, 18.7 of BT discuss the calculation of the unit cell. Bragg’s Law  2d(sin  ) = is known, and  is readily calculated in experimental set-up

4. Properties of diffracted beam Each diffracted beam is defined by amplitude, wavelength, and phase. Amplitude – measured by intensity of recorded spot Wavelength – set by x-ray source Phase – major problem in solving crystal structures

5. Solving the phase Max Perutz and John Kendrew pioneered molecular isomorphic replacement (MIR), which utilizes heavy atom derivatives of protein crystals to introduce new diffraction patterns. Typically use metals such as mercury or platinum. Multiwavelength Anomalous Diffraction (MAD) is extremely popular but requires synchrotron radiation in addition to a heavy metal derivative. The intensity differences obtained in the diffraction pattern using x-rays of different wavelengths can be used in a way similar to MIR. The sensitivity permitted by synchrotron radiation allows the use of lighter elements. The most tractable and useful method has proven to be incorporation of selenomethionine into expressed proteins.

6. Structural Genomic Consortia and HTS structure determination http://www.rcsb.org/pdb/strucgen.html#Worldwide http://www.stromix.com/

7. How can I tell if a crystal structure is insightful? Crystal structures are reported at a variety of resolutions 5 Angstroms – can make out secondary structures, but not individual groups of atoms 3 Angstroms – can trace alpha carbon backbone, but not sidechains 1.5 Angstroms – Good resolution R Factor – Each crystal structure will report this value, which corresponds to error associated with the model; In general, 0.2 or lower indicates a well-determined protein structure. B Factor – Temperature factor, should be 20 or less for good structures. Surface loops or terminal regions often have high B values due to flexibility, leading to disorder

8. Structure from Nuclear Magnetic Resonance Sample is placed in a strong magnetic field and exposed to radiofrequency radiation. Energy absorption is characteristic of the nuclei (H 1 or C 13 ), and its chemical environment. Allows structure determination under solution conditions Some limitation in size, but larger magnets helping http://www.nmrfam.wisc.edu/

9. The Future of Cell Biology?? http://www.pnas.org/cgi/reprint/97/26/14245.pdf Toward detecting and identifying macromolecules in a cellular context: Template matching applied to electron tomograms

10. Additional approaches to probe structure: 1)Fluorescence 2)Limited proteolysis 3)Circular dichroism (useful for secondary structure) 4)Deuterium exchange

11. Domains are revealed in protein structures Characterized by secondary structure content –All  –All  –  /  –  + 

12. Light-harvesting complex was an example of all 

13. Diversity in  structures Four helix bundle Seven membrane spanning regions in proteins involved in signal reception (ie. bacteriorhodopsin) Globin fold Different folds can affect #residues/turn

14. Packing  helices

15. Sequence/structure All a proteins begin to reveal sequence/structure relationship Coiled-coil proteins exhibit periodicity every seventh residue (heptad repeat); also seen in formation of dimers (GCN4) Observe hydrophobic moments in membrane proteins

16. Bacteriorhodopsin function

17. ~1/3 of all predicted proteins in a genome are membrane proteins

18. A different periodicity in  - structures

19. Common structures found in  structures Barrels Propellers Greek key Jelly roll (Contains one Greek key) Helix

20. Barrels – anti-parallel sheets

21. Anti-parallel structures exhibit every other amino acid periodicity

22. Propellers Variable number of propeller blades http://info.bio.cmu.edu/courses/03231/ProtStruc/b-props.htm

23. Propeller blade Ninety degree twist between first and fourth strand

24. Quaternary structure of neuraminidase

25. Looking for active sites

26. Greek key barrels Only n + 3 folds are observed

27. A topological examination of  - crystallin

28.  -crystallin has two domains with identical topology Protein evolution – motif duplication and fusion

29. Jelly roll motif Connections made over the end of the barrel

30. Another topology example

31. Parallel  -helix domains 3 aa in , 6 aa in turn. 18 aa/motif 9 aa repeat

32. Three sheet  -helix = Toblerone

33. Protein structures containing  and  Distinction between  /  and  +  –  /  - Mainly parallel beta sheets (beta-alpha- beta units) –  +  - Mainly antiparallel beta sheets (segregated alpha and beta regions)

34. 

35. Interspersed  and 

36. Generally, a tight hydrophobic core found in  barrels

37. How many folds are there? To date we know ~8000 protein structures Within this dataset, 450 folds are recognized Proteins have a common fold if they have the same major secondary structures in the same arrangement and with the same topological connections. http://scop.mrc-lmb.cam.ac.uk/scop/

38. How many non-folds are there? http://www.scripps.edu/news/press/013102. htmlhttp://www.scripps.edu/news/press/013102. html 30-40% of human genome encodes for “unstructured” native proteins

39. Think about Domains!