Python scripts for global fitting of T1-edited DEER data used in "Deconvoluting monomer- and dimer-specific distance distributions between spin labels in a monomer/dimer mixture using T1-edited DEER spectroscopy" by T. Schmidt, N. Kubatova and G.M. Clore, J. Am. Chem. Soc. 2024, doi:10.1021/jacs.4c03916

Double electron-electron resonance (DEER) EPR is a powerful tool in structural biology, providing distances between pairs of spin labels. When the sample consists of a mixture of oligomeric species (e.g. monomer and dimer) the question arises as to how to assign the peaks in the DEER-derived probability distance distribution to the individual species. Here we propose incorporating an EPR longitudinal electron relaxation (<i>T</i>₁) inversion recovery experiment within a DEER pulse sequence to resolve this problem. The apparent <i>T</i>₁ between dipolar coupled electron spins measured from the inversion recovery time (t_inv) dependence of the peak intensities in the <i>T</i>₁-edited DEER-derived probability <i>P</i>(<i>r</i>) distance distribution will be affected by the number of nitroxide labels attached to the biomolecule of interest, for example, two for a monomer and four for a dimer. We show that global fitting of all the <i>T</i>₁-edited DEER echo curves, recorded over a range of inversion recovery times, permits the deconvolution of distances between spin labels originating from monomeric (longer <i>T</i>₁) and dimeric (shorter <i>T</i>₁) species. This is especially useful when the trapping of spin labels in different conformational states during freezing gives rise to complex <i>P</i>(<i>r</i>) distance distributions. The utility of this approach is demonstrated for two systems, the β₁ adrenergic receptor and a construct of the huntingtin exon-1 protein fused to the immunoglobulin domain of protein G, both of which exist in a monomer-dimer equilibrium.

双电子-电子共振（Double electron-electron resonance, DEER）电子顺磁共振（electron paramagnetic resonance, EPR）是结构生物学中的强有力研究工具，可精准测定成对自旋标记物之间的距离。当样品为寡聚体混合物（如单体与二聚体）时，如何将DEER得到的概率距离分布中的峰归属至对应的单个物种，便成为一个关键科学问题。本文提出将EPR纵向电子弛豫（T₁）反转恢复实验整合至DEER脉冲序列中，以解决该难题。经T₁编辑的DEER得到的概率P(r)距离分布中，峰强度随反转恢复时间（t_inv）变化，由此测得的偶极耦合电子自旋间表观T₁，会受目标生物分子上连接的氮氧自由基标记物数量影响：例如单体对应2个标记物，二聚体则对应4个。研究表明，对一系列反转恢复时间下记录的所有经T₁编辑的DEER回波曲线进行全局拟合，可实现源自单体（T₁更长）与二聚体（T₁更短）物种的自旋标记物间距的解卷积。当冷冻过程中自旋标记物被捕获于不同构象状态，进而产生复杂的P(r)距离分布时，该方法尤为实用。本文通过两个体系验证了该方法的实用性：分别为β₁肾上腺素能受体，以及融合至蛋白G免疫球蛋白结构域的亨廷顿蛋白外显子1构建体，二者均存在单体-二聚体平衡。