This work has been supported by the excellence cluster &#34;The Hamburg Center for Ultrafast Imaging &#8211; Structure, Dynamics and Control of Matter at the Atomic Scale&#34; of the Deutsche Forschungsgemeinschaft and by the Helmholtz Virtual Institute &#34;Dynamic Pathways in Multidimensional Landscapes&#34;.

1. Description of the Experimental Setup
A schematic of the gas-phase molecular beam setup and deflector is shown in Figure 221. It consists of
<ol>
	<li>A pulsed Even-Lavie valve29 containing the molecular sample. Other pulsed molecular beam valves can be used equally well as long as a cold molecular beam (O(1 K)) is formed. The following parameters are specific for the employed Even-Lavie valve. In the experiments presented here the valve is operated at 20 Hz repetition rate with high backing pressures (helium at ~50 bar) and expanded into a vacuum chamber evacuated to &#60;10-6&#160;mbar.</li>
	<li>A molecular beam skimmer (2 mm diameter) is placed 22 cm downstream from the valve, collimating the molecular beam and leading to differential pumping conditions between the pulsed valve and the rest of the vacuum system.</li>
	<li>Immediately after the skimmer the molecules enter the electrostatic deflection device. This consists of a rod (radius 3.0 mm) and a trough (radius of curvature 3.2 mm), each 24 cm long. The vertical gap between the electrodes in the center&#160;of the device is 2.3 mm. A potential difference between 0-26 kV is applied between the rod and trough, producing a strong inhomogeneous electric field with a nearly constant field gradient30, as indicated in the inset of Figure 2.</li>
	<li>Directly after the deflector molecules enter the interaction region through a second&#160;skimmer, providing a further differential pumping stage.</li>
	<li>The interaction region (evacuated to pressures &#60;10-9 mbar) contains a standard Wiley-McLaren time-of-flight (TOF) setup. Molecules are ionized by focused laser pulses in the center of the extraction region, between the repeller and extractor electrodes. Produced ions are accelerated towards a multichannel plate (MCP) detector, where a mass spectrum is recorded.</li>
	<li>Laser pulses are derived from an Nd:YAG pumped dye laser, providing typical output wavelengths around 283 nm (indole experiments) or 272 nm (3-fluorophenol experiments) and pulse energies of a few mJ. Pulse durations are on the order of 10 nsec and pulses are focused with a f = 750 mm lens to a spot size of ~100 &#956;m in the interaction region.</li>
	<li>The timing sequence is controlled by a digital delay generator providing the master clock. This triggers the Nd:YAG laser (flash lamps and Q-switch), the pulsed valve, and the digitizer card used to record mass spectra.</li>
	<li>Mass spectra are recorded on a digitizer card, triggered at the same time as the laser Q-switch. Molecular beam densities are extracted from appropriate mass gates in the recorded time-of-flight spectra.</li>
</ol>
2. Production and Characterization of a Conformer Selected Molecular Beam
<ol>
	<li>A cold molecular beam of the target molecules is created via supersonic expansion and characterized using spatial (x, y directions) and temporal (z direction) profiling.&#160;</li>
	<li>Load the sample reservoir of the pulsed valve with the chemical sample. Dissolve solid samples in an appropriate solvent and place a few drops on a small piece of filter paper which is inserted into the sample cartridge. Place liquid samples directly on the filter paper.</li>
	<li>Produce the supersonic expansion, using a high-purity high-pressure backing gas. Adjust the temperature of the sample reservoir within the valve such that the partial pressure of the sample is approximately 10 mbar. 
	Note: For liquid samples typically no heating is necessary. The valve opening time depends on the exact model of pulsed valve used, for the experiments presented here the Even-Lavie valve is operated with an electric pulse duration of 10 &#956;sec.</li>
	<li>Characterize the produced molecular beam with the electrostatic deflector turned off. Set the ionization laser to a known wavelength for resonance-enhanced multiphoton ionization (REMPI) of a particular conformer of the sample. Record a temporal profile of the molecular beam pulse by monitoring the total parent ion yield on the MCP detector as a function of valve-laser delay.</li>
	<li>Fix the valve-laser delay at the position of maximum intensity for all subsequent measurements.</li>
	<li>Record a transverse spatial profile of the molecular beam by monitoring the total parent ion yield as a function of the y position of the laser focus. Do this by moving the focusing lens perpendicular to the laser propagation direction, such that the focus moves in the y direction relative to the molecular beam.</li>
	<li>Repeat the temporal and spatial profiling for all conformers of interest in the beam. 
	Note: These typically have distinct REMPI resonances, such that each conformer can be probed separately. In the absence of a deflection field however, the temporal and spatial profiles are identical for all conformers.</li>
	<li>Characterization of the deflected beam. Turn on the high voltage supply to the deflector and record spatial profiles for all isomers. These should now be deflected according to mass-to-dipole moment ratio. 
	Note: For species undergoing large deflections it may be necessary to move the skimmer immediately following the deflector to ensure good transmission of the deflected beam into the detection region.</li>
	<li>Conduct experiments on the conformer or size-selected part of the molecular beam by ensuring the interaction (e.g.&#160;a crossing laser beam) takes place within the part of the molecular beam containing only the species of interest.</li>
</ol>

The authors have nothing to disclose.

Throughout this manuscript, familiarity with ultra-high vacuum components, pulsed molecular beam valves and laser sources is assumed and the associated safety procedures should always be adhered to. Special care needs to be taken when handling the high voltage electrodes for the deflector. Their surfaces need to be polished to a high standard and must be absolutely clean to avoid arcing inside the vacuum chamber. Before first use the electrodes should be conditioned under vacuum. The voltage applied is slowly increased and the current throug...

Modern gas-phase molecular physics and physical chemistry experiments often use supersonic expansions of target molecules to produce rotationally cold molecular samples within a molecular beam. However, even at low rotational temperatures of 1 K, which can routinely be achieved using supersonic expansions, large molecules can still remain in multiple conformations within the beam1. Similarly the production of molecular clusters in a beam source does not result in a single species, but rather in the formation of a &#34;cluster soup&#34;, containing many different cluster stoichiometries, as well as remaining pure parent molecules. This makes the study of these systems with novel techniques such as imaging of molecular orbitals2, molecular-frame photoelectron angular distributions3-5 or electron6-10 and X-ray diffraction11-13 difficult, as these require pure, consistent, and homogenous samples in the gas-phase.
While several methodologies are now available to separate different conformers of charged species in the gas-phase (e.g. ion mobility drift tubes14,15) and charged clusters are easily separated by their mass-to-charge ratio, these techniques are not applicable to neutral species. We have recently demonstrated that these issues can be overcome with the use of an electrostatic deflection device16,17, allowing the separation of molecular conformers as well as clusters and the production of rotationally cold molecular beams.
The use of electrostatic deflection is a classic molecular beam technique, the origins of which go a long way back18,19. First ideas of utilizing electrostatic deflection for the separation of quantum states were introduced by Stern in 192620. While early experiments were conducted on small molecules at high temperatures, we demonstrate the application of this technique to large polar molecules and clusters at low temperatures16,21.
Polar molecules experience a force inside an inhomogenous electric field (E) due to the spatial differences in potential energy. This force<img fo:content-width="0.25in" src="/files/ftp_upload/51137/force.jpg" width="30px" /> is dependent on the effective dipole moment, &#956;eff, of the molecule and can be evaluated as
<img src="/files/ftp_upload/51137/51137eq1.jpg" /> (1) 
As different molecular conformers typically posses different dipole moments and differing numbers of solvent molecules within a cluster lead to different cluster masses and dipole moments, these species will experience a different acceleration in the presence of a strong inhomogeneous electric field. The resulting Stark effect force from an inhomogeneous electric field can therefore be used for the separation of conformers and quantum states22. This is indicated in Figure 1, showing the calculated Stark curves for the J = 0,1,2 rotational states of the cis and trans conformers of 3-fluorophenol, respectively. This leads to large differences in &#956;eff, as shown in Figures 1c and 1d, and hence a different acceleration is experienced by the two conformers in inhomogeneous electric fields. Therefore, an electrostatic deflection device can be used as a mass-to-dipole moment ratio (m/&#956;eff) separator, in analogy to a mass spectrometer acting as a mass-to-charge ratio (m/z) filter23.
Furthermore, these techniques allow the separation of rotational quantum states24,25. As the ground rotational states (blue curves in Figures 1a and 1b) exhibit the largest Stark shift, these will be deflected most and can be spatially separated from molecules in higher J states17. The coldest part of a molecular beam can therefore be selected, significantly aiding in many applications, such as alignment and orientation of target molecules17, 26-28.
In this contribution we show how an electrostatic deflection device can be used to spatially separate different species of large polar molecules and clusters. Example data is presented for the production of a pure beam of an individual conformer and of a solute-solvent cluster of well-defined size and ratio. Specifically we present data on 3-fluorophenol, where a pure beam containing only the trans conformer is produced, and on indole-water clusters, where the indole(H2O)1 cluster can be spatially separated from water, indole, indole(H2O)2 , etc.

<table><tbody><tr><td>Vacuum system</td><td>various, &lt;em&gt;e.g&lt;/em&gt;. Pfeiffer Vacuum, Varian, Edwards, Leybold</td><td></td><td></td></tr><tr><td>Dye laser system</td><td>various, &lt;em&gt;e.g&lt;/em&gt;. Coherent, Spectra Physics, Syrah, LIOP-TEC, Radiant Dyes&amp;hellip;</td><td></td><td></td></tr><tr><td>Pulsed valve</td><td>Even-Lavie</td><td></td><td></td></tr><tr><td>High voltage power supply</td><td>eg. FUG</td><td>HCP 14-20000</td><td></td></tr><tr><td>Deflector</td><td></td><td></td><td>Custom made</td></tr><tr><td>Time-of-flight spectrometer</td><td>Jordan TOF</td><td>C-677</td><td></td></tr><tr><td>TOF power supply</td><td>Jordan TOF</td><td>D-603</td><td></td></tr><tr><td>Focusing lens</td><td>Thorlabs</td><td>LA4745</td><td></td></tr><tr><td>Translation stage</td><td>&lt;em&gt;e.g&lt;/em&gt;. Vision Lasertechnik</td><td>8MT167-25</td><td></td></tr><tr><td>Digitizer</td><td>&lt;em&gt;e.g&lt;/em&gt;. Agilent</td><td>Acquiris DC440</td><td></td></tr><tr><td>Digital delay generator</td><td>Stanford Systems</td><td>SRS DG645</td><td></td></tr><tr><td>Molecular beam skimmer</td><td>Beam Dynamics Inc.</td><td></td><td>http://www.beamdynamicsinc.com/</td></tr></tbody></table>

spatial separation of molecular conformers and clusters

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.

The electrostatic deflection technique has been successfully applied to the separation of structural isomers16 and neutral clusters21, as well as the production of rotational quantum state selected molecular samples31. We demonstrate this with representative results for the separation of cis and trans conformers of 3-fluorophenol, and size selected indole(H2O)n clusters.
3-Fluorophenol conformers were separated in a molec...

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Spatial Separation of Molecular Conformers and Clusters

We present a technique that allows the spatial separation of different conformers or clusters present in a molecular beam. An electrostatic deflector is used to separate species by their mass-to-dipole moment ratio, leading to the production of gas-phase ensembles of a single conformer or cluster stoichiometry.

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold ...

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8.8K Views.  CFEL, DESY. We present a technique that allows the spatial separation of different conformers or clusters present in a molecular beam. An electrostatic deflector is used to separate species by their mass-to-dipole moment ratio, leading to the production of gas-phase ensembles of a single conformer or cluster stoichiometry.

Video: Spatial Separation of Molecular Conformers and Clusters

Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3, drug delivery4, improved hydrocarbon recovery5-7, and flowable electrodes for energy storage8. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius&#160;and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9.&#160;Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37.&#160;In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.

Confocal microscopy is used to image quiescent and flowing colloid-polymer mixtures, which are studied as model systems for attractive suspensions. Image analysis algorithms are used to calculate structural and dynamic metrics for the colloidal particles that measure changes due to geometric confinement.

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed ...

Chemistry

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality for: (1) The identification of related protein structure sets to user specified thresholds of similarity; (2) Their multiple alignment and structure superposition; (3) Sequence and structure conservation analysis; (4) Inter-conformer relationship mapping with principal component analysis, and (5) comparison of predicted internal dynamics via ensemble normal mode analysis. This integrated functionality provides a complete online workflow for investigating sequence-structure-dynamic relationships within protein families and superfamilies.

A protocol for the online investigation of protein sequence-structure-dynamics relationships using Bio3D-web is presented.

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Biochemistry

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

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Microcrystallography of Protein Crystals and In Cellulo Diffraction

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in their largest dimension, which used to represent a challenge. We present two alternative workflows for the structure determination of protein microcrystals by X-ray crystallography with a particular focus on crystals grown in vivo. The microcrystals are either extracted from cells by sonication and purified by differential centrifugation, or analyzed in cellulo after cell sorting by flow cytometry of crystal-containing cells. Optionally, purified crystals or crystal-containing cells are soaked in heavy atom solutions for experimental phasing. These samples are then prepared for diffraction experiments in a similar way by application onto a micromesh support and flash cooling in liquid nitrogen. We briefly describe and compare serial diffraction experiments of isolated microcrystals and crystal-containing cells using a microfocus synchrotron beamline to produce datasets suitable for phasing, model building and refinement.
These workflows are exemplified with crystals of the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produced by infection of insect cells with a recombinant baculovirus. In this case study, in cellulo analysis is more efficient than analysis of purified crystals and yields a structure in ~8 days from expression to refinement.

Microcrystallography of Protein Crystals and In Cellulo Diffraction

A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.

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Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their components. Therefore, the arrangements in the clusters have been precisely characterized, especially for metal complexes. Contrary to this, characterizations of molecular arrangements in supramolecular clusters composed of organic molecules are limited to a few cases. This is because construction of the supramolecular clusters, especially obtaining a series of the supramolecular clusters, is difficult due to low stability of non-covalent bonds compare to covalent bonds. From this viewpoint, utilization of organic salts is one of the most useful strategies. A series of the supramolecules could be constructed by combinations of a specific organic molecule with various counter ions. Especially, primary ammonium carboxylates are suitable as typical examples of supramolecules because various kinds of carboxylic acids and primary amines are commercially available, and it is easy to change their combinations. Previously, it was demonstrated that primary ammonium triphenylacetates using various kinds of primary amines specifically construct supramolecular clusters, which are composed of four ammoniums and four triphenylacetates assembled by charge-assisted hydrogen bonds, in crystals obtained from non-polar solvents. This study demonstrates an application of the specific construction of the supramolecular clusters as a strategy to conduct systematical symmetric study for clarification of correlations between molecular arrangements in supramolecules and kinds and numbers of their components. In the same way with binary salts composed of triphenylacetates and one kind of primary ammoniums, ternary organic salts composed of triphenylacetates and two kinds of ammoniums construct the supramolecular clusters, affording a series of the supramolecular clusters with various kinds and numbers of the components.

This article describes construction of a series of hydrogen-bonding supramolecular clusters in crystals using primary ammonium triphenylacetates, which are recrystallized from non-polar solvents. This selective construction of the supramolecular clusters leads to effective systematical symmetric studies about a correlation between the supramolecular clusters and their components.

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their ...

Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish between enantiomers (stereoisomers) of simple chiral species on the level of individual molecules. In this approach, a gaseous molecular jet of the sample expands into a vacuum chamber and intersects with femtosecond (fs) laser pulses. The high intensity of the pulses leads to fast multiple ionization, igniting a so-called Coulomb Explosion that produces several cationic (positively charged) fragments. An electrostatic field guides these cations onto time- and position-sensitive detectors. Similar to a time-of-flight mass spectrometer, the arrival time of each ion yields information on its mass. As a surplus, the electrostatic field is adjusted in a way that the emission direction and the kinetic energy after fragmentation lead to variations in the time-of-flight and in the impact position on the detector.
Each ion impact creates an electronic signal in the detector; this signal is treated by high-frequency electronics and recorded event by event by a computer. The registered data correspond to the impact times and positions. With these data, the energy and the emission direction of each fragment can be calculated. These values are related to structural properties of the molecule under investigation, i.e. to the bond lengths and relative positions of the atoms, allowing to determine molecule by molecule the handedness of simple chiral species and other isomeric features.

For small chiral species, Coulomb Explosion Imaging provides a new approach to determine the handedness of individual molecules.

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Computation of Atmospheric Concentrations of Molecular Clusters from ab initio Thermochemistry

The computational study of the formation and growth of atmospheric aerosols requires an accurate Gibbs free energy surface, which can be obtained from gas phase electronic structure and vibrational frequency calculations. These quantities are valid for those atmospheric clusters whose geometries correspond to a minimum on their potential energy surfaces. The Gibbs free energy of the minimum energy structure can be used to predict atmospheric concentrations of the cluster under a variety of conditions such as temperature and pressure. We present a computationally inexpensive procedure built on a genetic algorithm-based configurational sampling followed by a series of increasingly accurate screening calculations. The procedure starts by generating and evolving the geometries of a large set of configurations using semi-empirical models then refines the resulting unique structures at a series of high-level ab initio levels of theory. Finally, thermodynamic corrections are computed for the resulting set of minimum-energy structures and used to compute the Gibbs free energies of formation, equilibrium constants, and atmospheric concentrations. We present the application of this procedure to the study of hydrated glycine clusters under ambient conditions.

Computation of Atmospheric Concentrations of Molecular Clusters from ab initio Thermochemistry

The atmospheric concentrations of weakly bound molecular clusters can be computed from the thermochemical properties of low energy structures found through a multi-step configurational sampling methodology utilizing a genetic algorithm and semi-empirical and ab initio quantum chemistry.

The computational study of the formation and growth of atmospheric aerosols requires an accurate Gibbs free energy surface, which can be obtained from gas ...

Chemical Dimerization-Induced Protein Condensates on Telomeres

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E. coli dihydrofolate reductase (eDHFR), respectively. Fusion of Halo enzyme to a telomere protein anchors dimerizers to telomeres through covalent Halo ligand-enzyme binding. Binding of TMP to eDHFR recruits eDHFR-fused phase separating proteins to telomeres and induces condensate formation. Because TMP-eDHFR interaction is non-covalent, condensation can be reversed by using excess free TMP to compete with the dimerizer for eDHFR binding. An example of inducing promyelocytic leukemia (PML) nuclear body formation on telomeres and determining condensate growth, dissolution, localization and composition is shown. This method can be easily adapted to induce condensates at other genomic locations by fusing Halo to a protein that directly binds to the local chromatin or to dCas9 that is targeted to the genomic locus with a guide RNA. By offering the temporal resolution required for single cell live imaging while maintaining phase separation in a population of cells for biochemical assays, this method is suitable for probing both the formation and function of chromatin-associated condensates.

This protocol illustrates a chemically induced protein dimerization system to create condensates on chromatin.&#160; The formation of promyelocytic leukemia (PML) nuclear body on telomeres with chemical dimerizers is demonstrated. Droplet growth, dissolution, localization and composition are monitored with live cell imaging, immunofluorescence (IF) and fluorescence in situ hybridization (FISH).

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a ...

Biology

Molecular Orbital (MO) Theory

Source: Tamara M. Powers, Department of Chemistry, Texas A&#38;M University
This protocol serves as a guide in the synthesis of two metal complexes featuring the ligand 1,1&#39;-bis(diphenylphosphino)ferrocene (dppf): M(dppf)Cl2, where M = Ni or Pd. While both of these transition metal complexes are 4-coordinate, they exhibit different geometries at the metal center. Using molecular orbital (MO) theory in conjunction with 1H NMR and Evans method, we will determine the geometry of these two compounds.

Molecular Orbital MO Theory: Predicting Geometry of Transition Metal Complexes

Source: Tamara M. Powers, Department of Chemistry, Texas A&#38;M University
This protocol serves as a guide in the synthesis of two metal complexes ...

Spatial Separation of Molecular Conformers and Clusters

Summary

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Chapters in this video

Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time

Computation of Atmospheric Concentrations of Molecular Clusters from ab initio Thermochemistry

Chemical Dimerization-Induced Protein Condensates on Telomeres

Molecular Orbital (MO) Theory