The recent resurgence of ESR (electron spin resonance) in structural biology is in large part due to the development of distance measurements. This application was made possible by targeting of specific sites/domains by cysteine mutagenesis. Four techniques were developed to cover various distance ranges: exchange ESR for the very short distances (4-8 Å) (Miick et al. 1992) ; static and dynamic dipolar cw ESR (continuous wave ESR) for the 8-25 Å distance range (McHaourab et al. 1997; Rabenstein

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... and Shin 1995) ; and two pulsed methods: DEER (double electron electron resonance) (Milov et al. 1981 ) and DQC (double quantum coherence) (Borbat et al. 2002) for distances between 17 and 80 Å. The first two methods rely on line shape broadening and are thus limited to strong interactions (short distances). The pulsed methods extract weaker dipolar interactions from spin coherence and are thus sensitive to longer interspin distances. In addition, distances can be measured between the nitroxides and paramagnetic metals that enhance the relaxation of nitroxides (Budker et al. 1995; Voss et al. 1995) . All these techniques have been extensively tested and verified across the full distance range of sensitivity on model systems including organic biradicals (Jeschke et al. 2000), proteins (Sale et al. 2005) and DNA (Borbat et al. 2004; Schiemann et al. 2004). The use of extrinsic spin labels for distance measurements allows targeting of specific sites by side-directed spin labeling, which is of great advantage. However, the observed distances are between unpaired electrons at the end of long tethers (spin labels) that are attached to the molecular surface. To interpret these distances in terms of molecular structure, one needs to account for label length and conformation. Currently, the accuracy of the various ESR methods is of the order of 3 Å when label conformation is taken into account (Sale et al. 2005) . This chapter will focus entirely on one of the pulsed dipolar methods, DEER (double electron electron resonance). DEER, otherwise known as pulsed electron-electron resonance (PELDOR), was developed in Russia during the 1980s by Milov and collaborators (Milov et al. 1981 (Milov et al. , 1984 . Twenty years later Jeschke proposed a deadtime-free variant of the method, 4-pulse DEER, which, combined with commercially available instrumentation and advances in site-directed labeling, resulted in its resur-