Interpreting a Low Resolution Map of Human U1 snRNP Using Anomalous Scatterers

Chris Oubridge,Adelaine K.-W. Leung,Kiyoshi Nagai,Jade Li,Daniel A. Pomeranz Krummel

doi:10.1016/j.str.2009.05.009

Chris Oubridge, Adelaine K.-W. Leung + Show 3 more

Open Access

https://doi.org/10.1016/j.str.2009.05.009

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Abstract

SummaryWe recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 Å resolution electron density map. At 5–7 Å resolution, α helices and β sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 Å. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

Highlights

Most proteins in eukaryotic cells exist as components of large protein, RNA-protein, or DNA-protein complexes, which carry out important biological functions in an integrated manner (Gavin et al, 2002)
The functional core of human U1 snRNP consists of U1 snRNA and nine proteins
Ta6Br12 cluster coordinates and occupancies were refined in SHARP

Summary

Introduction

Most proteins in eukaryotic cells exist as components of large protein, RNA-protein, or DNA-protein complexes, which carry out important biological functions in an integrated manner (Gavin et al, 2002). Even low resolution diffraction data (5–7 A ) can provide essential insights into the functions of large macromolecular assemblies, provided one can obtain and interpret an electron density map (Ban et al, 1999; Clemons et al, 1999; Murakami et al, 2002; Bushnell et al, 2004). At such resolutions, a helices appear as tubular density (Muirhead and Perutz, 1963) and b sheets as flat density (Ban et al, 1999; Clemons et al, 1999). It may be possible to place a protein of known structure into the electron density map at this resolution, if its secondary structure is largely a-helical; it is, difficult to follow the polypeptide chain of a protein of unknown structure and determine its fold de novo

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