X-Ray Crystallography

  • Lauren E. Roth
  • F. Akif TezcanEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 766)


X-ray crystallography has been particularly important in the study of the enzyme nitrogenase, providing researchers with high-resolution structural models that have been essential to studying the enzyme’s unique metal clusters and nucleotide-binding modes and protein interactions. While several important nitrogenase structures have already been determined using X-ray crystallography, the technique still holds great potential for future significant discoveries involving reaction intermediates and redox states of the enzyme’s metal clusters. Thus, it is important to inform future nitrogenase researchers about the procedures for obtaining crystals of nitrogenase component proteins and their complexes and determining their structures. While nitrogenase component proteins from several bacteria have been crystallized, the majority of structures and those of highest resolution are of the nitrogenase proteins from the bacteria Azotobacter vinelandii. Therefore, the bulk of this chapter will focus on methods for crystallization and structure determination of nitrogenase component proteins from A. vinelandii.

Key words

Protein crystallography X-ray diffraction nitrogenase complexes 


  1. 1.
    Christiansen J, Dean DR, Seefeldt LC (2001) Mechanistic features of the Mo-containing nitrogenase. Annu Rev Plant Physiol Plant Mol Biol 52:269–295PubMedCrossRefGoogle Scholar
  2. 2.
    Burgess BK (1990) The Iron-Molybdenum cofactor of nitrogenase. Chem Rev 90:1377–1406CrossRefGoogle Scholar
  3. 3.
    Madden MS, Krezel AM, Allen RM et al (1992) Plausible structure of the iron-molybdenum cofactor of nitrogenase. Proc Natl Acad Sci USA 89:6487–6491PubMedCrossRefGoogle Scholar
  4. 4.
    Oliver ME, Hales BJ (1992) Structural information of the nitrogenase metal clusters deduced from paramagnetic interactions. J Am Chem Soc 114:10618–10623CrossRefGoogle Scholar
  5. 5.
    Georgiadias MM, Chakrabarti KP, Woo D et al (1992) Crystallographic structure of the nitrogenase Iron protein from Azotobacter vinelandii. Science 257:1653–1659CrossRefGoogle Scholar
  6. 6.
    Kim J, Rees DC (1992) Crystallographic structure and functional implications of the nitrogenase molybdenum-iron protein from Azotobacter vinelandii. Nature 360:553–560CrossRefGoogle Scholar
  7. 7.
    Roe SM, Gormal C, Smith BE et al (1997) Crystallization and preliminary X-ray studies of nitrogenase component 1 (the MoFe protein) from Klebsiella pneumonia. Acta Cryst D 53:227–228CrossRefGoogle Scholar
  8. 8.
    Mayer SM, Lawson DM, Gormal CA et al (1999) New insights into structure-function relationships in nitrogenase: a 1.6Å resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein. J Mol Biol 292:871–891PubMedCrossRefGoogle Scholar
  9. 9.
    Kim J, Woo D, Rees DC (1993) X-ray crystal structure of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum at 3.0-A resolution. Biochemistry 32:104–107Google Scholar
  10. 10.
    Schlessman JL, Woo D, Joshua-Tor L et al (1998) Conformational variability in structures of the nitrogenase iron proteins from Azotobacter vinelandii and Clostridium pasteurianum. J Mol Biol 280:669–685PubMedCrossRefGoogle Scholar
  11. 11.
    Chan MK, Kim J, Rees DC (1993) The nitrogenase FeMo-cofactor and P-cluster pair: 2.2 A resolution structures. Science 260:792–794PubMedCrossRefGoogle Scholar
  12. 12.
    Peters JW, Stowell MHB, Soltis SM et al (1997) Redox-dependent structural changes in the nitrogenase P-cluster. Biochemistry 36:1181–1187PubMedCrossRefGoogle Scholar
  13. 13.
    Einsle O, Tezcan FA, Andrade SLA et al (2002) Nitrogenase MoFe-protein at 1.16 Å resolution: a central ligand in the FeMo-cofactor. Science 297:1696–1700PubMedCrossRefGoogle Scholar
  14. 14.
    Jang SB, Seefeldt LC, Peters JW (2000) Insights into nucleotide signal transduction in nitrogenase: structure of an iron protein with MgADP bound. Biochemistry 39:14745–14752PubMedCrossRefGoogle Scholar
  15. 15.
    Jeong MS, Jang Se Bok (2004) Structural basis for the changes in redox potential in the nitrogenase Phe135Trp Fe protein with MgADP bound. Mol Cells 18:374–382PubMedGoogle Scholar
  16. 16.
    Jang SB, Jeong MS, Seefeldt LC et al (2004) Structural and biochemical implications of single amino acid substitutions in the nucleotide-dependent switch regions of the nitrogenase Fe protein from Azotobacter vinelandii. J Biol Inorg Chem 9:1028–1033PubMedCrossRefGoogle Scholar
  17. 17.
    Sen S, Igarashi R, Smith A et al (2004) A conformational mimic of the MgATP-bound “on state” of the nitrogenase iron protein. Biochemistry 43:1787–1797PubMedCrossRefGoogle Scholar
  18. 18.
    Sen S, Krishnakumar A, McClead J et al (2006) Insights into the role of nucleotide-dependent conformational change in nitrogenase catalysis: structural characterization of the nitrogenase Fe protein Leu127 deletion variant with bound MgATP. J Inorg Biochem 100:1041–1052PubMedCrossRefGoogle Scholar
  19. 19.
    Strop P, Takahara PM, Chiu H et al (2001) Crystal structure of the all-ferrous [4Fe-4S]0 form of the nitrogenase iron protein from Azotobacter vinelandii. Biochemistry 40:651–656PubMedCrossRefGoogle Scholar
  20. 20.
    Schindelin H, Kisker C, Schlessman JL et al (1997) Structure of ADP∙AlF4—stabilized nitrogenase complex and its implications for signal transduction. Nature 387:370–376PubMedCrossRefGoogle Scholar
  21. 21.
    Chiu H, Peters JW, Lanzilotta WN et al (2001) MgATP-bound and nucleotide-free structures of a nitrogenase protein complex between the Leu 127Δ-Fe-protein and the MoFe-protein. Biochemistry 40:641–650PubMedCrossRefGoogle Scholar
  22. 22.
    Tezcan FA, Kaiser JT, Mustafi D et al (2005) Nitrogenase complexes: multiple docking sites for a nucleotide switch protein. Science 309:1377–1380PubMedCrossRefGoogle Scholar
  23. 23.
    Sarma R, Barney BM, Keables S et al (2010) Insights into substrate binding at FeMo-cofactor in nitrogenase from the structure of an alpha-70Ile MoFe protein variant. J Inorg Biochem 104:385–389PubMedCrossRefGoogle Scholar
  24. 24.
    Sørlie M, Christiansen J, Lemon BJ et al (2001) Mechanistic features and structure of the nitrogenase α-Gln195 MoFe protein. Biochemistry 40:1540–1549PubMedCrossRefGoogle Scholar
  25. 25.
    Schmid B, Ribbe MW, Einsle O et al (2002) Structure of a cofactor-deficient nitrogenase MoFe protein. Science 296:352–356PubMedCrossRefGoogle Scholar
  26. 26.
    Mayer SM, Gormal CA, Smith BE et al (2002) Crystallographic analysis of the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae identifies citrate as a ligand to the molybdenum of iron molybdenum cofactor (FeMoco). J Biol Chem 277:35263–35266PubMedCrossRefGoogle Scholar
  27. 27.
    Jang SB, Seefeldt LC, Peters JW (2000) Modulating the midpoint potential of the [4Fe-4S] cluster of the nitrogenase Fe protein. Biochemistry 39:641–648PubMedCrossRefGoogle Scholar
  28. 28.
    Schmid B, Einsle O, Chiu H et al (2002) Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4--stabilized structure. Biochemistry 41:15557–15565PubMedCrossRefGoogle Scholar
  29. 29.
    Kim J, Rees DC (1992) Structural models for the metal centers in the nitrogenase molybdenum-iron protein. Science 257:1677–1682PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of CaliforniaSan Diego, La JollaUSA

Personalised recommendations