An isomorphous replacement method for efficient de novo phasing for serial femtosecond crystallography.

Tomohiko Okuda,Takaki Hatsui,Naoki Kajiyama,Masaki Yamamoto,Hideo Ago,Tomohiro Yamaguchi,So Iwata,Makina Yabashi,Atsushi Kodan,Toru Nakatsu,Michihiro Sugahara,Eiichi Mizohata,Changyong Song,Yasumasa Joti,Taichi Murai ,Mamoru Suzuki,Eriko Nango,Hiroaki Kato,Keiko Gomi,Takashi Kameshima,Keitaro Yamashita,Jaehyun Park,Dongqing Pan,Kensuke Tono

doi:10.1038/srep14017

Abstract

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) holds great potential for structure determination of challenging proteins that are not amenable to producing large well diffracting crystals. Efficient de novo phasing methods are highly demanding and as such most SFX structures have been determined by molecular replacement methods. Here we employed single isomorphous replacement with anomalous scattering (SIRAS) for phasing and demonstrate successful application to SFX de novo phasing. Only about 20,000 patterns in total were needed for SIRAS phasing while single wavelength anomalous dispersion (SAD) phasing was unsuccessful with more than 80,000 patterns of derivative crystals. We employed high energy X-rays from SACLA (12.6 keV) to take advantage of the large anomalous enhancement near the LIII absorption edge of Hg, which is one of the most widely used heavy atoms for phasing in conventional protein crystallography. Hard XFEL is of benefit for de novo phasing in the use of routinely used heavy atoms and high resolution data collection.

Highlights

We demonstrated that the single isomorphous replacement with anomalous scattering (SIRAS) method was successful for SFX de novo phasing with the indexed patterns of 10,792 native and 10,000 Hg derivative crystals whereas the SAD method was not www.nature.com/scientificreports/
The SAD method is contingent on the accurate measurement of the anomalous differences which are small (≲10% to F )
SIRAS phasing employed here benefited from both good isomorphism between crystals and the availability of high resolution data

Summary

Introduction

In this SIRAS phasing, we used ~20,000 indexed patterns with 10,000 each for derivative and native crystals. A notable reduction in the number of diffraction patterns required for successful phasing of SFX data by SIRAS is achieved compared to that needed for SAD phasing. Monte-Carlo integration yielded the mean SFX multiplicity of 222 at 1.5 Å resolution, when all indexed patterns were used.

Results

Conclusion