Structure determination of an integral membrane protein at room temperature from crystals in situ.

Danny Axford,Nien-Jen Hu,James Foadi,So Iwata,Yilmaz Alguel,Konstantinos Beis,Gwyndaf Evans,Hassanul Ghani Choudhury

doi:10.1107/s139900471500423x

Abstract

The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

Highlights

Membrane-protein structure determination routinely uses X-ray diffraction data recorded at cryogenic temperatures from a single crystal, requiring a significant investment of effort to grow samples of sufficient size to allow a complete data set to be recorded
We present a method to collect data from multiple in situ crystals of membrane proteins and to form a sufficiently complete data set from many partial data sets
A complete data set to 2.3 Aresolution was assembled from 63 partial data sets obtained by irradiating in situ 56 crystals of the membrane protein Haemophilus influenza TehA (HiTehA) distributed across a number

Summary

Introduction

Membrane-protein structure determination routinely uses X-ray diffraction data recorded at cryogenic temperatures from a single crystal, requiring a significant investment of effort to grow samples of sufficient size to allow a complete data set to be recorded. These two criteria have been driven by the typical nature of membrane-protein crystals: they are formed by limited crystal contacts, owing to a high solvent content and poor order, and are prone to non-isomorphism; these factors typically lead to weak diffraction (compared with most crystals of soluble proteins), requiring proportionally higher X-ray doses to allow measurement of high-resolution reflections. Membrane-protein crystal structure determination has been beyond the reach of room-temperature crystal diffraction measurements at synchrotron-radiation sources, principally owing to the significant primary and secondary

Methods

Results

Conclusion