Second Harmonic Generation Guided Raman Spectroscopy for Sensitive Detection of Polymorph Transitions Chowdhury, Azhad U., Ye, Dong Hye In: 2017. @article{noKey,
title = {Second Harmonic Generation Guided Raman Spectroscopy for Sensitive Detection of Polymorph Transitions},
author = {Chowdhury, Azhad U., Ye, Dong Hye},
url = {https://pubs.acs.org/doi/10.1021/acs.analchem.7b00431},
doi = {https://doi.org/10.1021/acs.analchem.7b00431},
year = {2017},
date = {2017-01-01},
abstract = {Second harmonic generation (SHG) was integrated with Raman spectroscopy for the
analysis of pharmaceutical materials. Particulate formulations of clopidogrel bisulphate were
prepared in two crystal forms (Form I and Form II). Image analysis approaches enable
automated identification of particles by bright field imaging, followed by classification by SHG.
Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with
99.95% confidence in a total measurement time of ~10 ms per particle. Complementary
measurements by Raman and synchrotron XRD are in excellent agreement with the
classifications made by SHG, with measurement times of ~1 minute and several seconds per
particle, respectively. Coupling these capabilities with at-line monitoring may enable real-time
feedback for reaction monitoring during pharmaceutical production to favor the more
bioavailable but metastable Form I with limits of detection in the ppm regime.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Second harmonic generation (SHG) was integrated with Raman spectroscopy for the
analysis of pharmaceutical materials. Particulate formulations of clopidogrel bisulphate were
prepared in two crystal forms (Form I and Form II). Image analysis approaches enable
automated identification of particles by bright field imaging, followed by classification by SHG.
Quantitative SHG microscopy enabled discrimination of crystal form on a per particle basis with
99.95% confidence in a total measurement time of ~10 ms per particle. Complementary
measurements by Raman and synchrotron XRD are in excellent agreement with the
classifications made by SHG, with measurement times of ~1 minute and several seconds per
particle, respectively. Coupling these capabilities with at-line monitoring may enable real-time
feedback for reaction monitoring during pharmaceutical production to favor the more
bioavailable but metastable Form I with limits of detection in the ppm regime. |
Nonlinear Optical Characterization of Membrane Protein Microcrystals and Nanocrystals Newman, Justin A., Simpson, Garth J. In: 2016. @article{noKey,
title = {Nonlinear Optical Characterization of Membrane Protein Microcrystals and Nanocrystals},
author = {Newman, Justin A., Simpson, Garth J.},
url = {https://link.springer.com/chapter/10.1007%2F978-3-319-35072-1_7},
doi = {https://doi.org/10.1007/978-3-319-35072-1_7},
year = {2016},
date = {2016-01-01},
abstract = {Nonlinear optical methods such as second harmonic generation (SHG) and
two-photon excited UV fluorescence (TPE-UVF) imaging are promising
approaches to address bottlenecks in the membrane protein structure
determination pipeline. The general principles of SHG and TPE-UVF are
discussed here along with instrument design considerations. Comparisons
to conventional methods in high throughput crystallization condition
screening and crystal quality assessment prior to X-ray diffraction are also
discussed.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Nonlinear optical methods such as second harmonic generation (SHG) and
two-photon excited UV fluorescence (TPE-UVF) imaging are promising
approaches to address bottlenecks in the membrane protein structure
determination pipeline. The general principles of SHG and TPE-UVF are
discussed here along with instrument design considerations. Comparisons
to conventional methods in high throughput crystallization condition
screening and crystal quality assessment prior to X-ray diffraction are also
discussed. |
Characterization of Protein Nanocrystals Based on the Reversibility of Crystallization Dörner, Katerina, Garcia, Jose M. Martin- In: 2016. @article{noKey,
title = {Characterization of Protein Nanocrystals Based on the Reversibility of Crystallization},
author = {Dörner, Katerina, Garcia, Jose M. Martin-},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5649632/},
doi = {https://doi.org/10.1021/acs.cgd.6b00384},
year = {2016},
date = {2016-01-01},
abstract = {A new approach is described to screen for protein nanocrystals based on the reversibility of crystallization. Methods to characterize nanocrystals are in strong need to facilitate sample preparation for serial femtosecond X-ray nanocrystallography (SFX). SFX enables protein structure determination by collecting X-ray diffraction from nano- and microcrystals using a free electron laser. This technique is especially valuable for challenging proteins as for example membrane proteins and is in general a powerful method to overcome the radiation damage problem and to perform time-resolved structure analysis. Nanocrystal growth cannot be monitored with common methods used in protein crystallography, as the resolution of bright field microscopy is not sufficient. A high-performance method to screen for nanocrystals is second order nonlinear imaging of chiral crystals (SONICC). However, the high cost prevents its use in every laboratory, and some protein nanocrystals may be �invisible� to SONICC. In this work using a crystallization robot and a common imaging system precipitation comprised of nanocrystals and precipitation caused by aggregated protein can be distinguished.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
A new approach is described to screen for protein nanocrystals based on the reversibility of crystallization. Methods to characterize nanocrystals are in strong need to facilitate sample preparation for serial femtosecond X-ray nanocrystallography (SFX). SFX enables protein structure determination by collecting X-ray diffraction from nano- and microcrystals using a free electron laser. This technique is especially valuable for challenging proteins as for example membrane proteins and is in general a powerful method to overcome the radiation damage problem and to perform time-resolved structure analysis. Nanocrystal growth cannot be monitored with common methods used in protein crystallography, as the resolution of bright field microscopy is not sufficient. A high-performance method to screen for nanocrystals is second order nonlinear imaging of chiral crystals (SONICC). However, the high cost prevents its use in every laboratory, and some protein nanocrystals may be �invisible� to SONICC. In this work using a crystallization robot and a common imaging system precipitation comprised of nanocrystals and precipitation caused by aggregated protein can be distinguished. |
Protein Crystallization in an Actuated Microfluidic Nanowell Device Abdallah, Bahige G., Chowdhury, Shatabdi Roy- In: 2016. @article{noKey,
title = {Protein Crystallization in an Actuated Microfluidic Nanowell Device},
author = {Abdallah, Bahige G., Chowdhury, Shatabdi Roy-},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036579/},
doi = {https://doi.org/10.1021/acs.cgd.5b01748},
year = {2016},
date = {2016-01-01},
abstract = {Protein crystallization is a major bottleneck of structure determination by X-ray crystallography, hampering the process by years in some cases. Numerous matrix screening trials using significant amounts of protein are often applied, while a systematic approach with phase diagram determination is prohibited for many proteins that can only be expressed in small amounts. Here, we demonstrate a microfluidic nanowell device implementing protein crystallization and phase diagram screening using nanoscale volumes of protein solution per trial. The device is made with cost-effective materials and is completely automated for efficient and economical experimentation. In the developed device, 170 trials can be realized with unique concentrations of protein and precipitant established by gradient generation and isolated by elastomeric valving for crystallization incubation. Moreover, this device can be further downscaled to smaller nanowell volumes and larger scale integration. The device was calibrated using a fluorescent dye and compared to a numerical model where concentrations of each trial can be quantified to establish crystallization phase diagrams. Using this device, we successfully crystallized lysozyme and C-phycocyanin, as visualized by compatible crystal imaging techniques such as bright-field microscopy, UV fluorescence, and second-order nonlinear imaging of chiral crystals. Concentrations yielding observed crystal formation were quantified and used to determine regions of the crystallization phase space for both proteins. Low sample consumption and compatibility with a variety of proteins and imaging techniques make this device a powerful tool for systematic crystallization studies.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Protein crystallization is a major bottleneck of structure determination by X-ray crystallography, hampering the process by years in some cases. Numerous matrix screening trials using significant amounts of protein are often applied, while a systematic approach with phase diagram determination is prohibited for many proteins that can only be expressed in small amounts. Here, we demonstrate a microfluidic nanowell device implementing protein crystallization and phase diagram screening using nanoscale volumes of protein solution per trial. The device is made with cost-effective materials and is completely automated for efficient and economical experimentation. In the developed device, 170 trials can be realized with unique concentrations of protein and precipitant established by gradient generation and isolated by elastomeric valving for crystallization incubation. Moreover, this device can be further downscaled to smaller nanowell volumes and larger scale integration. The device was calibrated using a fluorescent dye and compared to a numerical model where concentrations of each trial can be quantified to establish crystallization phase diagrams. Using this device, we successfully crystallized lysozyme and C-phycocyanin, as visualized by compatible crystal imaging techniques such as bright-field microscopy, UV fluorescence, and second-order nonlinear imaging of chiral crystals. Concentrations yielding observed crystal formation were quantified and used to determine regions of the crystallization phase space for both proteins. Low sample consumption and compatibility with a variety of proteins and imaging techniques make this device a powerful tool for systematic crystallization studies. |
X-ray Transparent Microfluidics for Protein Rystallization and Biomineralization Opathalage, Achini In: 2016. @article{noKey,
title = {X-ray Transparent Microfluidics for Protein Rystallization and Biomineralization},
author = {Opathalage, Achini},
url = {https://pdfs.semanticscholar.org/ac78/5adf2f188296dbff23315d8764ce8ad747a7.pdf},
doi = {undefined},
year = {2016},
date = {2016-01-01},
abstract = {X-ray transparent Microfluidics for Protein Crystallization and Biomineralization A dissertation presented to the Faculty of the Graduate School of Arts and Sciences of Brandeis University, Waltham, Massachusetts by Achini Opathalage Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions.In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 �. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
X-ray transparent Microfluidics for Protein Crystallization and Biomineralization A dissertation presented to the Faculty of the Graduate School of Arts and Sciences of Brandeis University, Waltham, Massachusetts by Achini Opathalage Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions.In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 �. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3. |
The detection and subsequent volume optimization of biological nanocrystals Luft, Joseph R., Wolfley, Jennifer R. In: 2015. @article{noKey,
title = {The detection and subsequent volume optimization of biological nanocrystals},
author = {Luft, Joseph R., Wolfley, Jennifer R.},
url = {https://aca.scitation.org/doi/full/10.1063/1.4921199},
doi = {https://doi.org/10.1063/1.4921199},
year = {2015},
date = {2015-01-01},
abstract = {Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography. Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging. The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions. It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions. A crystal volume optimization method is described and associated with a phase diagram for crystallization.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography. Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging. The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions. It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions. A crystal volume optimization method is described and associated with a phase diagram for crystallization. |
Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser Wu, Wenting, Nogly, Przemyslaw In: 2015. @article{noKey,
title = {Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser},
author = {Wu, Wenting, Nogly, Przemyslaw},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498706/},
doi = {https://doi.org/10.1107/S2053230X15009966},
year = {2015},
date = {2015-01-01},
abstract = {Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup. |
Intercalating dyes for enhanced contrast in second-harmonic generation imaging of protein crystals Newman, Justin A., Scarborough, Nicole M. In: 2015. @article{noKey,
title = {Intercalating dyes for enhanced contrast in second-harmonic generation imaging of protein crystals},
author = {Newman, Justin A., Scarborough, Nicole M.},
url = {http://scripts.iucr.org/cgi-bin/paper?S1399004715008287},
doi = {https://dx.doi.org/10.1107%2FS1399004715008287},
year = {2015},
date = {2015-01-01},
abstract = {The second-harmonic generation (SHG) activity of protein crystals was found to be enhanced by up to ~1000-fold by the intercalation of SHG phores within the crystal lattice. Unlike the intercalation of fluorophores, the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates. The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice. In addition, the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response. Kinetics studies indicate that intercalation arises over a timeframe of several minutes in lysozyme, with detectable enhancements within seconds. These results provide a potential means to increase the overall diversity of protein crystals and crystal sizes amenable to characterization by SHG microscopy.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
The second-harmonic generation (SHG) activity of protein crystals was found to be enhanced by up to ~1000-fold by the intercalation of SHG phores within the crystal lattice. Unlike the intercalation of fluorophores, the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates. The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice. In addition, the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response. Kinetics studies indicate that intercalation arises over a timeframe of several minutes in lysozyme, with detectable enhancements within seconds. These results provide a potential means to increase the overall diversity of protein crystals and crystal sizes amenable to characterization by SHG microscopy. |
Reliably distinguishing protein nanocrystals from amorphous precipitate by means of depolarized dynamic light scattering Schubert, Robin, Meyer, Arne In: 2015. @article{noKey,
title = {Reliably distinguishing protein nanocrystals from amorphous precipitate by means of depolarized dynamic light scattering},
author = {Schubert, Robin, Meyer, Arne},
url = {http://scripts.iucr.org/cgi-bin/paper?S1600576715014740},
doi = {https://doi.org/10.1107/S1600576715014740},
year = {2015},
date = {2015-01-01},
abstract = {Crystallization of biological macromolecules such as proteins implies several prerequisites, for example, the presence of one or more initial nuclei, sufficient amounts of the crystallizing substance and the chemical potential to provide the free energy needed to force the process. The initiation of a crystallization process itself is a stochastic event, forming symmetrically assembled nuclei over kinetically preferred protein-dense liquid clusters. The presence of a spatial repetitive orientation of macromolecules in the early stages of the crystallization process has so far proved undetectable. However, early identification of the occurrences of unit cells is the key to nanocrystal detection. The optical properties of a crystal lattice offer a potential signal with which to detect whether a transition from disordered to ordered particles occurs, one that has so far not been tested in nanocrystalline applications. The ability of a lattice to depolarize laser light depends on the different refractive indices along different crystal axes. In this study a unique experimental setup is used to detect nanocrystal formation by application of depolarized scattered light. The results demonstrate the successful detection of nano-sized protein crystals at early stages of crystal growth, allowing an effective differentiation between protein-dense liquid cluster formation and ordered nanocrystals. The results are further verified by complementary methods like X-ray powder diffraction, second harmonic generation, ultraviolet two-photon excited fluorescence and scanning electron microscopy.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Crystallization of biological macromolecules such as proteins implies several prerequisites, for example, the presence of one or more initial nuclei, sufficient amounts of the crystallizing substance and the chemical potential to provide the free energy needed to force the process. The initiation of a crystallization process itself is a stochastic event, forming symmetrically assembled nuclei over kinetically preferred protein-dense liquid clusters. The presence of a spatial repetitive orientation of macromolecules in the early stages of the crystallization process has so far proved undetectable. However, early identification of the occurrences of unit cells is the key to nanocrystal detection. The optical properties of a crystal lattice offer a potential signal with which to detect whether a transition from disordered to ordered particles occurs, one that has so far not been tested in nanocrystalline applications. The ability of a lattice to depolarize laser light depends on the different refractive indices along different crystal axes. In this study a unique experimental setup is used to detect nanocrystal formation by application of depolarized scattered light. The results demonstrate the successful detection of nano-sized protein crystals at early stages of crystal growth, allowing an effective differentiation between protein-dense liquid cluster formation and ordered nanocrystals. The results are further verified by complementary methods like X-ray powder diffraction, second harmonic generation, ultraviolet two-photon excited fluorescence and scanning electron microscopy. |
Microseed matrix screening for optimization in protein crystallization: what have we learned? D’Arcy, Allan, Bergfors, Terese In: 2014. @article{noKey,
title = {Microseed matrix screening for optimization in protein crystallization: what have we learned?},
author = {D’Arcy, Allan, Bergfors, Terese},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157405/},
doi = {https://doi.org/10.1107/S2053230X14015507},
year = {2014},
date = {2014-01-01},
abstract = {Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time- and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time- and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems. |
Characterization of salt interferences in second-harmonic generation detection of protein crystals Closser, R. G., Gualtieri, E. J. In: 2013. @article{noKey,
title = {Characterization of salt interferences in second-harmonic generation detection of protein crystals},
author = {Closser, R. G., Gualtieri, E. J.},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831302/},
doi = {https://doi.org/10.1107/S0021889813027581},
year = {2013},
date = {2013-01-01},
abstract = {Studies were undertaken to assess the merits and limitations of second-harmonic generation (SHG) for the selective detection of protein and polypeptide crystal formation, focusing on the potential for false positives from SHG-active salts present in crystallization media. The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples. Out of 19 salts investigated, six produced significant background SHG and 15 of the 96 wells of a sparse-matrix screen produced SHG upon solvent evaporation. SHG-active salts include phosphates, hydrated sulfates, formates and tartrates, while chlorides, acetates and anhydrous sulfates resulted in no detectable SHG activity. The identified SHG-active salts produced a range of signal intensities spanning nearly three orders of magnitude. However, even the weakest SHG-active salt produced signals that were several orders of magnitude greater than those produced by typical protein crystals. In general, SHG-active salts were identifiable through characteristically strong SHG and negligible two-photon-excited ultraviolet fluorescence (TPE-UVF). Exceptions included trials containing either potassium dihydrogen phosphate or ammonium formate, which produced particularly strong SHG, but with residual weak TPE-UVF signals that could potentially complicate discrimination in crystallization experiments using these precipitants.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Studies were undertaken to assess the merits and limitations of second-harmonic generation (SHG) for the selective detection of protein and polypeptide crystal formation, focusing on the potential for false positives from SHG-active salts present in crystallization media. The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples. Out of 19 salts investigated, six produced significant background SHG and 15 of the 96 wells of a sparse-matrix screen produced SHG upon solvent evaporation. SHG-active salts include phosphates, hydrated sulfates, formates and tartrates, while chlorides, acetates and anhydrous sulfates resulted in no detectable SHG activity. The identified SHG-active salts produced a range of signal intensities spanning nearly three orders of magnitude. However, even the weakest SHG-active salt produced signals that were several orders of magnitude greater than those produced by typical protein crystals. In general, SHG-active salts were identifiable through characteristically strong SHG and negligible two-photon-excited ultraviolet fluorescence (TPE-UVF). Exceptions included trials containing either potassium dihydrogen phosphate or ammonium formate, which produced particularly strong SHG, but with residual weak TPE-UVF signals that could potentially complicate discrimination in crystallization experiments using these precipitants. |
Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel Abdallah, Bahige G., Kupitz, Christopher In: 2013. @article{noKey,
title = {Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel},
author = {Abdallah, Bahige G., Kupitz, Christopher},
url = {http://www.ncbi.nlm.nih.gov/pmc/articles/3940344/},
doi = {https://doi.org/10.1021/nn402515q},
year = {2013},
date = {2013-01-01},
abstract = {Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these �difficult to crystallize� proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these �difficult to crystallize� proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. |
Dielectrophoretic sorting of membrane protein nanocrystals Abdallah, Bahige G., Chao, Tzu-Chiao In: 2013. @article{noKey,
title = {Dielectrophoretic sorting of membrane protein nanocrystals},
author = {Abdallah, Bahige G., Chao, Tzu-Chiao},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894612/},
doi = {https://doi.org/10.1021/nn403760q},
year = {2013},
date = {2013-01-01},
abstract = {Structure elucidation of large membrane protein complexes still comprises a considerable challenge yet is a key factor in drug development and disease combat. Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals, thus overcoming the experimental riddle faced in traditional crystallography methods. Here, we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis. We demonstrate the excellent sorting power of this new approach with numerical simulations of selected sub-micrometer beads in excellent agreement with experimental observations. Crystals from batch crystallization broths of the huge membrane protein complex photosystem I were sorted without further treatment, resulting in a high degree of monodispersity and crystallinity in the ~ 100 nm size range. Microfluidic integration, continuous sorting, and nanometer-sized crystal fractions make this method ideal for direct coupling to femtosecond nanocrystallography.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Structure elucidation of large membrane protein complexes still comprises a considerable challenge yet is a key factor in drug development and disease combat. Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals, thus overcoming the experimental riddle faced in traditional crystallography methods. Here, we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis. We demonstrate the excellent sorting power of this new approach with numerical simulations of selected sub-micrometer beads in excellent agreement with experimental observations. Crystals from batch crystallization broths of the huge membrane protein complex photosystem I were sorted without further treatment, resulting in a high degree of monodispersity and crystallinity in the ~ 100 nm size range. Microfluidic integration, continuous sorting, and nanometer-sized crystal fractions make this method ideal for direct coupling to femtosecond nanocrystallography. |
Membrane protein structure determination by electron crystallography Belandia, Iban Ubarretxena-, Stokes, David L. In: 2013. @article{noKey,
title = {Membrane protein structure determination by electron crystallography},
author = {Belandia, Iban Ubarretxena-, Stokes, David L.},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423591/},
doi = {https://doi.org/10.1016/j.sbi.2012.04.003},
year = {2013},
date = {2013-01-01},
abstract = {During the past year, electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer. From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope. There have also been key developments in software, and in molecular replacement and phase extension methods designed to facilitate the process of structure determination.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
During the past year, electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer. From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope. There have also been key developments in software, and in molecular replacement and phase extension methods designed to facilitate the process of structure determination. |
Towards protein-crystal centering using second-harmonic generation (SHG) microscopy Kissick, David J., Dettmar, Christopher M. In: 2013. @article{noKey,
title = {Towards protein-crystal centering using second-harmonic generation (SHG) microscopy},
author = {Kissick, David J., Dettmar, Christopher M.},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3640472/},
doi = {https://doi.org/10.1107/S0907444913002746},
year = {2013},
date = {2013-01-01},
abstract = {The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-�ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using �2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-�ray minibeam. SHG imaging was found to provide about 2 �m spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-�ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using �2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-�ray minibeam. SHG imaging was found to provide about 2 �m spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed. |
Second-order nonlinear optical imaging of chiral crystals Kissick, David J., Wanapun, Debbie In: 2012. @article{noKey,
title = {Second-order nonlinear optical imaging of chiral crystals},
author = {Kissick, David J., Wanapun, Debbie},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345893/},
doi = {https://doi.org/10.1146/annurev.anchem.111808.073722},
year = {2012},
date = {2012-01-01},
abstract = {Second-order nonlinear optical imaging of chiral crystals (SONICC) is an emerging technique for crystal imaging and characterization. We provide a brief overview of the origin of second harmonic generation signals in SONICC and discuss recent studies using SONICC for biological applications. Given that they provide near-complete suppression of any background, SONICC images can be used to determine the presence or absence of protein crystals through both manual inspection and automated analysis. Because SONICC creates high-resolution images, nucleation and growth kinetics can also be observed. SONICC can detect metastable, homochiral crystalline forms of amino acids crystallizing from racemic solutions, which confirms Ostwald�s rule of stages for crystal growth. SONICC�s selectivity, based on order, and sensitivity, based on background suppression, make it a promising technique for numerous fields concerned with chiral crystal formation.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Second-order nonlinear optical imaging of chiral crystals (SONICC) is an emerging technique for crystal imaging and characterization. We provide a brief overview of the origin of second harmonic generation signals in SONICC and discuss recent studies using SONICC for biological applications. Given that they provide near-complete suppression of any background, SONICC images can be used to determine the presence or absence of protein crystals through both manual inspection and automated analysis. Because SONICC creates high-resolution images, nucleation and growth kinetics can also be observed. SONICC can detect metastable, homochiral crystalline forms of amino acids crystallizing from racemic solutions, which confirms Ostwald�s rule of stages for crystal growth. SONICC�s selectivity, based on order, and sensitivity, based on background suppression, make it a promising technique for numerous fields concerned with chiral crystal formation. |
Screening of Protein Crystallization Trials by Second Order Nonlinear Optical Imaging of Chiral Crystals (SONICC) Haupert, Levi, Simpson, Garth In: 2011. @article{noKey,
title = {Screening of Protein Crystallization Trials by Second Order Nonlinear Optical Imaging of Chiral Crystals (SONICC)},
author = {Haupert, Levi, Simpson, Garth},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264792/},
doi = {https://doi.org/10.1016/j.ymeth.2011.11.003},
year = {2011},
date = {2011-01-01},
abstract = {Second order nonlinear optical imaging of chiral crystals (SONICC) is a promising new method for the sensitive and selective detection of protein crystals. Relevant general principles of second harmonic generation, which underpins SONICC, are reviewed. Instrumentation and methods for SONICC measurements are described and critically assessed in terms of performance trade-offs. Potential origins of false-positives and false-negatives are also discussed.},
keywords = {SONICC},
pubstate = {published},
tppubtype = {article}
}
Second order nonlinear optical imaging of chiral crystals (SONICC) is a promising new method for the sensitive and selective detection of protein crystals. Relevant general principles of second harmonic generation, which underpins SONICC, are reviewed. Instrumentation and methods for SONICC measurements are described and critically assessed in terms of performance trade-offs. Potential origins of false-positives and false-negatives are also discussed. |
