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Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.

ABSTRACT: Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3?Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1?ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1?mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.

SUBMITTER: Nogly P

PROVIDER: S-EPMC4996941 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.

Nogly Przemyslaw P Panneels Valerie V Nelson Garrett G Gati Cornelius C Kimura Tetsunari T Milne Christopher C Milathianaki Despina D Kubo Minoru M Wu Wenting W Conrad Chelsie C Coe Jesse J Bean Richard R Zhao Yun Y Båth Petra P Dods Robert R Harimoorthy Rajiv R Beyerlein Kenneth R KR Rheinberger Jan J James Daniel D DePonte Daniel D Li Chufeng C Sala Leonardo L Williams Garth J GJ Hunter Mark S MS Koglin Jason E JE Berntsen Peter P Nango Eriko E Iwata So S Chapman Henry N HN Fromme Petra P Frank Matthias M Abela Rafael R Boutet Sébastien S Barty Anton A White Thomas A TA Weierstall Uwe U Spence John J Neutze Richard R Schertler Gebhard G Standfuss Jörg J

Nature communications 20160822

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M sta ...[more]

PMID: 27545823

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Project description:Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

Dataset Information

Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.

Publications

Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.

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