Matching Items (4)

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Serial femtosecond crystallography datasets from G protein-coupled receptors

Description

We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic

We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH[subscript 2], the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data.

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Created

Date Created
  • 2016-08-01

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Serial crystallography at synchrotrons and X-ray lasers

Description

Recently we have seen rapid progress in the serial crystallography (SC) method at X-ray free-electron lasers (XFELs). Injection of thousands of protein microcrystals into the ∼10[superscript 12] photons of few-femtosecond

Recently we have seen rapid progress in the serial crystallography (SC) method at X-ray free-electron lasers (XFELs). Injection of thousands of protein microcrystals into the ∼10[superscript 12] photons of few-femtosecond XFEL pulses has allowed the structure determination of crystals grown in vivo, or of submicron size, and from challenging targets such as membrane proteins. For time-resolved studies, the small crystal size allows for rapid diffusive saturation in mix-and-inject analysis of biochemical reactions, and full optical saturation of the sample by a pump laser in studies of light-driven proteins. The ability to outrun most radiation damage avoids the need for sample cooling and its artifacts, allowing studies of molecular machines at work in their correct room-temperature thermal bath or a controlled chemical environment.

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Created

Date Created
  • 2017-03

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A novel inert crystal delivery medium for serial femtosecond crystallography

Description

Serial femtosecond crystallography (SFX) has opened a new era in crystallo­graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron

Serial femtosecond crystallography (SFX) has opened a new era in crystallo­graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

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Created

Date Created
  • 2015-06-30

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

Description

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

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.

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Created

Date Created
  • 2016-08-22