Modelling multi-protein complexes using PELDOR distance measurements for rigid body minimisation experiments using XPLOR-NIH

Colin M Hammond,Tom Owen-Hughes,David G Norman

doi:10.1016/j.ymeth.2014.10.028

Colin M Hammond, Tom Owen-Hughes + Show 1 more

Open Access

https://doi.org/10.1016/j.ymeth.2014.10.028

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Journal: Methods	Publication Date: Nov 18, 2014
Citations: 6	License type: cc-by

Affiliation: University of Dundee

Abstract

Crystallographic and NMR approaches have provided a wealth of structural information about protein domains. However, often these domains are found as components of larger multi domain polypeptides or complexes. Orienting domains within such contexts can provide powerful new insight into their function. The combination of site specific spin labelling and Pulsed Electron Double Resonance (PELDOR) provide a means of obtaining structural measurements that can be used to generate models describing how such domains are oriented. Here we describe a pipeline for modelling the location of thio-reactive nitroxyl spin locations to engineered sties on the histone chaperone Vps75. We then use a combination of experimentally determined measurements and symmetry constraints to model the orientation in which homodimers of Vps75 associate to form homotetramers using the XPLOR-NIH platform. This provides a working example of how PELDOR measurements can be used to generate a structural model.

Highlights

To date there have been nearly 100,000 structures deposited in the protein data bank (PDB)
The protocol focusses on a Abbreviations: Cys, cysteine; Glu, glutamic acid; Tyr, tyrosine; Vacuolar Protein Sorting 75 (Vps75), vacuolar protein sorting protein 75
E56R1 refers to Vps75 in which Glu 56 which has been mutated to Cys and cross-reacted with MTSL (Fig. 1A)

Summary

Introduction

To date there have been nearly 100,000 structures deposited in the protein data bank (PDB). This rich source of structural biological information is utilised by many laboratories to gain functional insights. A procedure is described which utilises Pulsed Electron Double Resonance (PELDOR) [alternatively referred to as double electron–electron resonance (DEER)] distance measurements as restraints to dock PDB structures together. This procedure can be extrapolated to utilise other sources of distance information, such as residue specific crosslinking information obtained from cross-linking MS/MS experiments. The protocol focusses on a Abbreviations: Cys, cysteine; Glu, glutamic acid; Tyr, tyrosine; Vps, vacuolar protein sorting protein 75

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Conclusion