NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM₄A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.

使用 NOE 衍生的距离限制进行 NMR 结构计算需要对主链和侧链共振进行大量分配，这对于大型或复杂蛋白质来说通常很难或不可能获得。赝接触位移 (PCS) 在 NMR 蛋白质结构计算中也发挥着重要作用，通常用于增强现有的结构信息（主要是 NOE 衍生的信息）。使用 PCS 的现有细化协议通常要么需要大量侧链分配，要么通过其他实验限制进行补充。在这里，我们提出了一种自动迭代程序来执行骨架蛋白质结构精化，仅需要有限量的骨架酰胺 PCS。来自起始同源模型的已知结构特征（在本例中是重复蛋白的模块）被构建成支架，随后通过实验 PCS 进行完善。该方法产生可靠的指标，可以对其进行监控以判断性能。我们将其应用到侧链分配几乎不可能的系统中，设计了犰狳重复蛋白（dArmRP），并计算了 YM ₄ A（包含四个序列相同内部模块的 dArmRP）的溶液 NMR 结构，获得了对单个序列的高度收敛。结构。我们建议，当从其他技术（例如 X 射线晶体学）获知近似折叠时，这种方法特别有用，同时避免了由于晶体堆积等原因造成的固有伪影。