Matching Items (10)
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Description

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

ContributorsIshchenko, Andrii (Author) / Cherezov, Vadim (Author) / Liu, Wei (Author) / College of Liberal Arts and Sciences (Contributor)
Created2016-09-20
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Description

The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser

The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

ContributorsZhang, Xianjun (Author) / Zhao, Fei (Author) / Wu, Yiran (Author) / Yang, Jun (Author) / Han, Gye Won (Author) / Zhao, Suwen (Author) / Ishchenko, Andrii (Author) / Ye, Lintao (Author) / Lin, Xi (Author) / Ding, Kang (Author) / Dharmarajan, Venkatasubramaniam (Author) / Griffin, Patrick R. (Author) / Gati, Cornelius (Author) / Nelson, Garrett (Author) / Hunter, Mark S. (Author) / Hanson, Michael A. (Author) / Cherezov, Vadim (Author) / Stevens, Raymond C. (Author) / Tan, Wenfu (Author) / Tao, Houchao (Author) / Xu, Fei (Author) / College of Liberal Arts and Sciences (Contributor)
Created2017-05-17
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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 cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an

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-NH2, 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.

ContributorsWhite, Thomas A. (Author) / Barty, Anton (Author) / Liu, Wei (Author) / Ishchenko, Andrii (Author) / Zhang, Haitao (Author) / Gati, Cornelius (Author) / Zatsepin, Nadia (Author) / Basu, Shibom (Author) / Oberthur, Dominik (Author) / Metz, Markus (Author) / Beyerlein, Kenneth R. (Author) / Yoon, Chun Hong (Author) / Yefanov, Oleksandr M. (Author) / James, Daniel (Author) / Wang, Dingjie (Author) / Messerschmidt, Marc (Author) / Koglin, Jason E. (Author) / Boutet, Sebastien (Author) / Weierstall, Uwe (Author) / Cherezov, Vadim (Author) / College of Liberal Arts and Sciences (Contributor)
Created2016-08-01
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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 the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement.

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.
ContributorsNogly, Przemyslaw (Author) / Panneels, Valerie (Author) / Nelson, Garrett (Author) / Gati, Cornelius (Author) / Kimura, Tetsunari (Author) / Milne, Christopher (Author) / Milathianaki, Despina (Author) / Kubo, Minoru (Author) / Wu, Wenting (Author) / Conrad, Chelsie (Author) / Coe, Jesse (Author) / Bean, Richard (Author) / Zhao, Yun (Author) / Bath, Petra (Author) / Dods, Robert (Author) / Harimoorthy, Rajiv (Author) / Beyerlein, Kenneth R. (Author) / Rheinberger, Jan (Author) / James, Daniel (Author) / Deponte, Daniel (Author) / Li, Chufeng (Author) / Sala, Leonardo (Author) / Williams, Garth J. (Author) / Hunter, Mark S. (Author) / Koglin, Jason E. (Author) / Berntsen, Peter (Author) / Nango, Eriko (Author) / Iwata, So (Author) / Chapman, Henry N. (Author) / Fromme, Petra (Author) / Frank, Matthias (Author) / Abela, Rafael (Author) / Boutet, Sebastien (Author) / Barty, Anton (Author) / White, Thomas A. (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Neutze, Richard (Author) / Schertler, Gebhard (Author) / Standfuss, Jorg (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / School of Molecular Sciences (Contributor)
Created2016-08-22
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Description
Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5–20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A[subscript 2A] adenosine receptor (A[subscript 2A]AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A[subscript 2A]AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A[subscript 2A]AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5–20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.
ContributorsMartin Garcia, Jose Manuel (Author) / Conrad, Chelsie (Author) / Nelson, Garrett (Author) / Stander, Natasha (Author) / Zatsepin, Nadia (Author) / Zook, James (Author) / Zhu, Lan (Author) / Geiger, James (Author) / Chun, Eugene (Author) / Kissick, David (Author) / Hilgart, Mark C. (Author) / Ogata, Craig (Author) / Ishchenko, Andrii (Author) / Nagaratnam, Nirupa (Author) / Roy Chowdhury, Shatabdi (Author) / Coe, Jesse (Author) / Subramanian, Ganesh (Author) / Schaffer, Alexander (Author) / James, Daniel (Author) / Ketwala, Gihan (Author) / Venugopalan, Nagarajan (Author) / Xu, Shenglan (Author) / Corcoran, Stephen (Author) / Ferguson, Dale (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Cherezov, Vadim (Author) / Fromme, Petra (Author) / Fischetti, Robert F. (Author) / Liu, Wei (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2017-05-24
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Description
Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of “diffraction-before-destruction.” However, de novo structure factor phase determination using XFELs

Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of “diffraction-before-destruction.” However, de novo structure factor phase determination using XFELs has been difficult so far. We demonstrate the ability to solve the crystallographic phase problem for SFX data collected with an XFEL using the anomalous signal from native sulfur atoms, leading to a bias-free room temperature structure of the human A[subscript 2A] adenosine receptor at 1.9 Å resolution. The advancement was made possible by recent improvements in SFX data analysis and the design of injectors and delivery media for streaming hydrated microcrystals. This general method should accelerate structural studies of novel difficult-to-crystallize macromolecules and their complexes.
ContributorsBatyuk, Alexander (Author) / Galli, Lorenzo (Author) / Ishchenko, Andrii (Author) / Han, Gye Won (Author) / Gati, Cornelius (Author) / Popov, Petr A. (Author) / Lee, Ming-Yue (Author) / Stauch, Benjamin (Author) / White, Thomas A. (Author) / Barty, Anton (Author) / Aquila, Andrew (Author) / Hunter, Mark S. (Author) / Liang, Mengning (Author) / Boutet, Sebastien (Author) / Pu, Mengchen (Author) / Liu, Zhi-jie (Author) / Nelson, Garrett (Author) / James, Daniel (Author) / Li, Chufeng (Author) / Zhao, Yun (Author) / Spence, John (Author) / Liu, Wei (Author) / Fromme, Petra (Author) / Katritch, Vsevolod (Author) / Weierstall, Uwe (Author) / Stevens, Raymond C. (Author) / Cherezov, Vadim (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / School of Molecular Sciences (Contributor)
Created2016-09-23
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Description
Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.
ContributorsFromme, Raimund (Author) / Ishchenko, Andrii (Author) / Metz, Markus (Author) / Roy Chowdhury, Shatabdi (Author) / Basu, Shibom (Author) / Boutet, Sebastien (Author) / Fromme, Petra (Author) / White, Thomas A. (Author) / Barty, Anton (Author) / Spence, John (Author) / Weierstall, Uwe (Author) / Liu, Wei (Author) / Cherezov, Vadim (Author) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2015-08-04
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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 XFEL pulses has allowed the structure determination of crystals grown in vivo, or of submicron size, and from challenging targets

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.
ContributorsStandfuss, Jorg (Author) / Spence, John (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2017-03
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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 laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption.

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.
ContributorsConrad, Chelsie (Author) / Basu, Shibom (Author) / James, Daniel (Author) / Wang, Dingjie (Author) / Schaffer, Alexander (Author) / Roy Chowdhury, Shatabdi (Author) / Zatsepin, Nadia (Author) / Aquila, Andrew (Author) / Coe, Jesse (Author) / Gati, Cornelius (Author) / Hunter, Mark S. (Author) / Koglin, Jason E. (Author) / Kupitz, Christopher (Author) / Nelson, Garrett (Author) / Subramanian, Ganesh (Author) / White, Thomas A. (Author) / Zhao, Yun (Author) / Zook, James (Author) / Boutet, Sebastien (Author) / Cherezov, Vadim (Author) / Spence, John (Author) / Fromme, Raimund (Author) / Weierstall, Uwe (Author) / Fromme, Petra (Author) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor)
Created2015-06-30
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Description
Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to

Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.
ContributorsZhou, X. Edward (Author) / Gao, Xiang (Author) / Barty, Anton (Author) / Kang, Yanyong (Author) / He, Yuanzheng (Author) / Liu, Wei (Author) / Ishchenko, Andrii (Author) / White, Thomas A. (Author) / Yefanov, Oleksandr (Author) / Han, Gye Won (Author) / Xu, Qingping (Author) / de Waal, Parker W. (Author) / Suino-Powell, Kelly M. (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Wang, Meitian (Author) / Li, Dianfan (Author) / Caffrey, Martin (Author) / Chapman, Henry N. (Author) / Spence, John (Author) / Fromme, Petra (Author) / Weierstall, Uwe (Author) / Stevens, Raymond C. (Author) / Cherezov, Vadim (Author) / Melcher, Karsten (Author) / Xu, H. Eric (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2016-04-12