powerpoint presentation on x ray crystallography

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X-ray Crystallography

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X-ray Crystallography is a scientific method used to determine the arrangement of atoms of a crystalline solid in three dimensional space. This technique takes advantage of the interatomic spacing of most crystalline solids by employing them as a diffraction gradient for x-ray light, which has wavelengths on the order of 1 angstrom (10 -8 cm).

Introduction

In 1895, Wilhelm Rontgen discovered x- rays. The nature of x- rays, whether they were particles or electromagnetic radiation, was a topic of debate until 1912. If the wave idea was correct, researchers knew that the wavelength of this light would need to be on the order of 1 Angstrom (A) (10 -8 cm). Diffraction and measurement of such small wavelengths would require a gradient with spacing on the same order of magnitude as the light.

In 1912, Max von Laue, at the University of Munich in Germany, postulated that atoms in a crystal lattice had a regular, periodic structure with interatomic distances on the order of 1 A. Without having any evidence to support his claim on the periodic arrangements of atoms in a lattice, he further postulated that the crystalline structure can be used to diffract x-rays, much like a gradient in an infrared spectrometer can diffract infrared light. His postulate was based on the following assumptions: the atomic lattice of a crystal is periodic, x- rays are electromagnetic radiation, and the interatomic distance of a crystal are on the same order of magnitude as x- ray light. Laue's predictions were confirmed when two researchers: Friedrich and Knipping, successfully photographed the diffraction pattern associated with the x-ray radiation of crystalline \(CuSO_4 \cdot 5H_2O\). The science of x-ray crystallography was born.

The arrangement of the atoms needs to be in an ordered, periodic structure in order for them to diffract the x-ray beams. A series of mathematical calculations is then used to produce a diffraction pattern that is characteristic to the particular arrangement of atoms in that crystal. X-ray crystallography remains to this day the primary tool used by researchers in characterizing the structure and bonding of organometallic compounds.

Diffraction

Diffraction is a phenomena that occurs when light encounters an obstacle. The waves of light can either bend around the obstacle, or in the case of a slit, can travel through the slits. The resulting diffraction pattern will show areas of constructive interference, where two waves interact in phase, and destructive interference, where two waves interact out of phase. Calculation of the phase difference can be explained by examining Figure 1 below.

In the figure below, two parallel waves, BD and AH are striking a gradient at an angle \(θ_o\). The incident wave BD travels farther than AH by a distance of CD before reaching the gradient. The scattered wave (depicted below the gradient) HF, travels father than the scattered wave DE by a distance of HG. So the total path difference between path AHGF and BCDE is CD - HG. To observe a wave of high intensity (one created through constructive interference), the difference CD - HG must equal to an integer number of wavelengths to be observed at the angle psi, \(CD - HG = n\lambda\), where \(\lambda\) is the wavelength of the light. Applying some basic trigonometric properties, the following two equations can be shown about the lines:

\[CD = x \cos(θ o) \nonumber \]

\[HG = x \cos (θ) \nonumber \]

where \(x\) is the distance between the points where the diffraction repeats. Combining the two equations,

Bragg's Law

Diffraction of an x-ray beam, occurs when the light interacts with the electron cloud surrounding the atoms of the crystalline solid. Due to the periodic crystalline structure of a solid, it is possible to describe it as a series of planes with an equal interplaner distance. As an x-ray's beam hits the surface of the crystal at an angle ?, some of the light will be diffracted at that same angle away from the solid (Figure 2). The remainder of the light will travel into the crystal and some of that light will interact with the second plane of atoms. Some of the light will be diffracted at an angle \(theta\), and the remainder will travel deeper into the solid. This process will repeat for the many planes in the crystal. The x-ray beams travel different pathlengths before hitting the various planes of the crystal, so after diffraction, the beams will interact constructively only if the path length difference is equal to an integer number of wavelengths (just like in the normal diffraction case above). In the figure below, the difference in path lengths of the beam striking the first plane and the beam striking the second plane is equal to BG + GF. So, the two diffracted beams will constructively interfere (be in phase) only if \(BG + GF = n \lambda\). Basic trigonometry will tell us that the two segments are equal to one another with the interplaner distance times the sine of the angle \(\theta\). So we get:

\[ BG = BC = d \sin \theta \label{1} \]

\[ 2d \sin \theta = n \lambda \label{2} \]

This equation is known as Bragg's Law, named after W. H. Bragg and his son, W. L. Bragg; who discovered this geometric relationship in 1912. {C}{C}Bragg's Law relates the distance between two planes in a crystal and the angle of reflection to the x-ray wavelength. The x-rays that are diffracted off the crystal have to be in-phase in order to signal. Only certain angles that satisfy the following condition will register:

\[ \sin \theta = \dfrac{n \lambda}{2d} \label{3} \]

For historical reasons, the resulting diffraction spectrum is represented as intensity vs. \(2θ\).

Instrument Components

The main components of an x-ray instrument are similar to those of many optical spectroscopic instruments. These include a source, a device to select and restrict the wavelengths used for measurement, a holder for the sample, a detector, and a signal converter and readout. However, for x-ray diffraction; only a source, sample holder, and signal converter/readout are required.

x-ray tubes provides a means for generating x-ray radiation in most analytical instruments. An evacuated tube houses a tungsten filament which acts as a cathode opposite to a much larger, water cooled anode made of copper with a metal plate on it. The metal plate can be made of any of the following metals: chromium, tungsten, copper, rhodium, silver, cobalt, and iron. A high voltage is passed through the filament and high energy electrons are produced. The machine needs some way of controlling the intensity and wavelength of the resulting light. The intensity of the light can be controlled by adjusting the amount of current passing through the filament; essentially acting as a temperature control. The wavelength of the light is controlled by setting the proper accelerating voltage of the electrons. The voltage placed across the system will determine the energy of the electrons traveling towards the anode. X-rays are produced when the electrons hit the target metal. Because the energy of light is inversely proportional to wavelength (\(E=hc=h(1/\lambda\)), controlling the energy, controls the wavelength of the x-ray beam.

X-ray Filter

Monochromators and filters are used to produce monochromatic x-ray light. This narrow wavelength range is essential for diffraction calculations. For instance, a zirconium filter can be used to cut out unwanted wavelengths from a molybdenum metal target (see figure 4). The molybdenum target will produce x-rays with two wavelengths. A zirconium filter can be used to absorb the unwanted emission with wavelength K β , while allowing the desired wavelength, K α to pass through.

Needle Sample Holder

The sample holder for an x-ray diffraction unit is simply a needle that holds the crystal in place while the x-ray diffractometer takes readings.

Signal Converter

In x-ray diffraction, the detector is a transducer that counts the number of photons that collide into it. This photon counter gives a digital readout in number of photons per unit time. Below is a figure of a typical x-ray diffraction unit with all of the parts labeled.

Fourier Transform

In mathematics, a Fourier transform is an operation that converts one real function into another. In the case of FTIR, a Fourier transform is applied to a function in the time domain to convert it into the frequency domain. One way of thinking about this is to draw the example of music by writing it down on a sheet of paper. Each note is in a so-called "sheet" domain. These same notes can also be expressed by playing them. The process of playing the notes can be thought of as converting the notes from the "sheet" domain into the "sound" domain. Each note played represents exactly what is on the paper just in a different way. This is precisely what the Fourier transform process is doing to the collected data of an x-ray diffraction. This is done in order to determine the electron density around the crystalline atoms in real space. The following equations can be used to determine the electrons' position:

\[p(x,y,z) = \sum_h \sum_k \sum_l F(hkl) e ^{-2\pi i (hx+ky+lz)} \label{1A} \]

\[ \int _0^1 \int _0^1 \int _0^1 p(x,y,z) e ^{2\pi i (hx+ky+lz)} dx\;dy\;dz \label{2B} \]

\[F(q) = | F(q) | e^{i \phi(q)} \label{3C} \]

where \(p(xyz)\) is the electron density function, and \(F(hkl)\) is the electron density function in real space. Equation 1 represents the Fourier expansion of the electron density function. To solve for \(F(hkl)\), the equation 1 needs to be evaluated over all values of h, k, and l, resulting in Equation 2. The resulting function \(F(hkl)\) is generally expressed as a complex number (as seen in equation 3 above) with \(| F(q)|\) representing the magnitude of the function and \(\phi\) representing the phase.

Crystallization

In order to run an x-ray diffraction experiment, one must first obtain a crystal. In organometallic chemistry, a reaction might work but when no crystals form, it is impossible to characterize the products. Crystals are grown by slowly cooling a supersaturated solution. Such a solution can be made by heating a solution to decrease the amount of solvent present and to increase the solubility of the desired compound in the solvent. Once made, the solution must be cooled gradually. Rapid temperature change will cause the compound to crash out of solution, trapping solvent and impurities within the newly formed matrix. Cooling continues as a seed crystal forms. This crystal is a point where solute can deposit out of the solution and into the solid phase. Solutions are generally placed into a freezer (-78 ºC) in order to ensure all of the compound has crystallized. One way to ensure gradual cooling in a -78 ºC freezer is to place the container housing the compound into a beaker of ethanol. The ethanol will act as a temperature buffer, ensuring a slow decrease in the temperature gradient between the flask and the freezer. Once crystals are grown, it is imperative that they remain cold as any addition of energy will cause a disruption of the crystal lattice, which will yield bad diffraction data. The result of an organometallic chromium compound crystallization can be seen below.

Mounting the Crystal

Due to the air-sensitivity of most organometallic compounds, crystals must be transported in a highly viscous organic compound called paratone oil (Figure \(\PageIndex{7}\)). Crystals are abstracted from their respective Schlenks by dabbing the end of a spatula with the paratone oil and then sticking the crystal onto the oil. Although there might be some exposure of the compounds to air and water, crystals can withstand more exposure than solution (of the preserved protein) before degrading. On top of serving to protect the crystal, the paratone oil also serves as the glue to bind the crystal to the needle.

Rotating Crystal Method

To describe the periodic, three dimensional nature of crystals, the Laue equations are employed:

\[ a(\cos \theta_o – \cos \theta) = h\lambda \label{eq1} \]

\[b(\cos \theta_o – \cos \theta) = k\lambda \label{eq2} \]

\[c(\cos \theta_o – \cos \theta) = l\lambda \label{eq3} \]

where \(a\), \(b\), and \(c\) are the three axes of the unit cell, \(θ_o\), \(o\), \(?o\) are the angles of incident radiation, and ?, ?, ? are the angles of the diffracted radiation. A diffraction signal (constructive interference) will arise when \(h\), \(k\), and \(l\) are integer values. The rotating crystal method employs these equations. X-ray radiation is shown onto a crystal as it rotates around one of its unit cell axis. The beam strikes the crystal at a 90 degree angle. Using equation 1 above, we see that if \(\theta_o\) is 90 degrees, then \(\cos \theta_o = 0\). For the equation to hold true, we can set h=0, granted that \(\theta= 90^o\). The above three equations will be satisfied at various points as the crystal rotates. This gives rise to a diffraction pattern (shown in the image below as multiple h values). The cylindrical film is then unwrapped and developed. The following equation can be used to determine the length axis around which the crystal was rotated:

\[ a = \dfrac{ch \lambda}{\sin \tan^{-1} (y/r} \nonumber \]

where \(a\) is the length of the axis, y is the distance from \(h=0\) to the \(h\) of interest, \(r\) is the radius of the firm, and ? is the wavelength of the x-ray radiation used. The first length can be determined with ease, but the other two require far more work, including remounting the crystal so that it rotates around that particular axis.

X-ray Crystallography of Proteins

The crystals that form are frozen in liquid nitrogen and taken to the synchrotron which is a highly powered tunable x-ray source. They are mounted on a goniometer and hit with a beam of x-rays. Data is collected as the crystal is rotated through a series of angles. The angle depends on the symmetry of the crystal.

Proteins are among the many biological molecules that are used for x-ray Crystallography studies. They are involved in many pathways in biology, often catalyzing reactions by increasing the reaction rate. Most scientists use x-ray Crystallography to solve the structures of protein and to determine functions of residues, interactions with substrates, and interactions with other proteins or nucleic acids. Proteins can be co - crystallized with these substrates, or they may be soaked into the crystal after crystallization.

Protein Crystallization

Proteins will solidify into crystals under certain conditions. These conditions are usually made up of salts, buffers, and precipitating agents. This is often the hardest step in x-ray crystallography. Hundreds of conditions varying the salts, pH, buffer, and precipitating agents are combined with the protein in order to crystallize the protein under the right conditions. This is done using 96 well plates; each well containing a different condition and crystals; which form over the course of days, weeks, or even months. The pictures below are crystals of APS Kinase D63N from Penicillium chrysogenum taken at the Chemistry building at UC Davis after crystals formed over a period of a week.

• Skoog, D . A.; Holler, F. J.; Stanley R. C.; Principles of Instrumental Analysis; Thomson Brooks/Cole: Belmont CA, 2007.
• Sands, D. E.; Introduction to Crystallography; Dover Publications, Inc.; New York, 1975
• Drenth, Jan. Principles of Protein x-ray Crystallography, 3rd edition. 2007, Springer Science + Business Media, LLC. pg. 14.
• Rhodes, Gale. Crystallography Made Crystal Clear, 3rd edition. 2006, Elsevier Inc. pg. 33, 55 - 57.
• Actual experimentation done of APS Kinase D63N Penicillium Chrysogenum.

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The Advanced Photon Source a U.S. Department of Energy Office of Science User Facility

Introduction to crystallography.

Filming ultrafast molecular motions in single crystal

U nderstanding the behavior of matter is crucial for advancing scientific fields like biology, chemistry, and materials science. X-ray crystallography has been instrumental in this pursuit, allowing scientists to determine molecular structures with precision.

In traditional X-ray crystallography experiments, a single crystal is exposed to X-rays multiple times to obtain diffraction signals. This poses a problem, where the sample has its structure altered or damaged by X-ray exposure.

In recent years, advances in technology have allowed for the development of "time-resolved serial femtosecond crystallography" (TR-SFX). In serial crystallography, a crystal is exposed to X-rays only once, which allows for the measurement of the sample in the best possible state where the crystal is not damaged by X-rays. This is then combined with the popular time-resolved technique, which allows the structural changes of molecules in crystals to be followed in real time during a reaction.

However, TR-SFX so far has only been limited to the study of protein samples. If the usage of TR-SFX can be extended to non-protein samples, it will unlock opportunities to investigate real-time motion across a wider range of materials, encompassing those crucial for semiconductors and batteries.

For the first time, researchers led by Director IHEE Hyotcherl of the Center for Advanced Reaction Dynamics within the Institute for Basic Science (IBS) have applied TR-SFX to a system other than proteins. The work has been published in Nature Chemistry .

The material they chose was a sample called porous coordination network–224(Fe), PCN–224(Fe), to demonstrate the feasibility of serial crystallography at the molecular level, allowing them to observe molecular motion in real time with atomic resolution.

The sample consists of carbon monoxide (CO) adsorbed onto iron porphyrin (Fe porphyrin) derivatives and zirconium (Zr) clusters repeated in a metal–organic framework.

The reason why TR-SFX was previously limited to only studying protein samples was that much higher standards are required for evaluating the structures of non-protein samples. Hence, the IBS team had to greatly improve the specification of the crystallography in order to meet these high criteria.

The team's setup revealed the crystal structure at a total of 33 time points ranging from 100 femtoseconds to 3 nanoseconds (10 -9 seconds). This is an advance over previous TR-SFX studies of the proteins, which typically report crystal structures at only about 10 time points. This substantial increase in temporal resolution, nearly three times greater than previous studies on proteins, allowed for a more accurate representation of structural changes over a long period of time.

When PCN–224(Fe) is irradiated with light, the CO adsorbed on the Fe porphyrin is dissociated, initiating a cascade of structural changes. Using the improved TR-SFX, researchers were able to observe these structural changes with unprecedented detail—with a femtosecond time resolution of 10 -15 seconds and an atomic resolution of 10 -10 meters (or angstroms).

They were able to identify three different pathways of structural change: doming, the movement of iron atoms in iron porphyrins out of the porphyrin plane; phonon mode of zirconium and iron atoms; and random vibrational motion with increasing temperature.

With this study, the researchers have shown that it is possible to apply TR-SFX measurements to chemical systems, an important step forward in demonstrating the practicality of the technique.

The study marks a major milestone for the scientific community as it is the first time molecular behavior has been observed in real-time using serial crystallography. By using TR-SFX, a technique that provides high spatiotemporal resolution, the team was able to capture minute structural changes in solid-state molecules in real time.

Director Ihee of the Center for Advanced Molecular Reaction Dynamics said, "Since the technical advances and analytical methods proposed in this study can be widely used to observe many other crystalline phase reactions of various molecular systems, this research not only opens new horizons in the field of molecular structure research but also has endless applications in future scientific discoveries."

More information: Dynamic 3D structures of a metal–organic framework captured with femtosecond serial crystallography, Nature Chemistry (2024). DOI: 10.1038/s41557-024-01460-w

Provided by Institute for Basic Science

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X-ray crystallography - PowerPoint PPT Presentation

X-ray crystallography

Don't get image of individual atoms. x-ray diffraction. works thanks to bragg equation ... x-ray crystallography. nl=2dsinq n is the 'order' ... – powerpoint ppt presentation.

• Atoms are 1-5Å apart so how do we see this?
• Between 0.1 and 10Å (1Å 0.1 nm)
• Atoms cause scattering
• Diffraction coherent scattering of waves off a periodic arrangement of matter
• Vacuum environment
• Source of electrons
• Large accelerating voltage
• Target metal
• Tungsten filament as source of e-
• E- sent to target metal (usually Cu)
• X-rays generated off Cu
• Higher voltage, faster e-, more energy
• X-ray generation
• Continuous spectra (white radiation)
• Electrons hit target surface, loose energy, stop
• NO change of target electron configuration
• Removed by filtering
• Characteristic radiation
• Electrons interact with target electron configuration
• Fingerprint of target metal
• Bombarding electrons dislodge electron from target
• K-shell or L-shell
• If vacancy in K-shell filled from L-shell, K??radiation
• If vacancy if K-shell filled from M-shell, K? radiation
• K? radiation has more energy than K? radiation
• Quantized energy and characteristic of metal target
• Characteristic radiation more intense than continuous
• Want Ka radiation to go to sample so need to filter everything else
• Photographically
• Electronic detectors
• Result see planes of atoms and what orientation planes are in
• Dont get image of individual atoms
• Works thanks to Bragg Equation
• X-rays diffract at specific angles based on spacing of atomic planes
• Destructive and constructive interference of waves
• Bragg Equation
• nl2dsinq n is the order
• As soon as the crystal is rotated, the beam ceases (This is diffraction, not reflection)
• Only get diffraction at certain angles!
• Relation between l and d and q

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Dalton Transactions

Aggregation-induced enhanced fluorescence emission of chiral zn(ii) complexes coordinated by schiff-base type binaphthyl ligands.

A pair of novel chiral Zn(II) complexes coordinated by Schiff-base type ligands derived from BINOL (1,1’-bi-2-naphthol), R -/ S -Zn , were synthesized. X-ray crystallography revealed the presence of two crystallographically independent complexes; one has a distorted trigonal-bipyramidal structure coordinated by two binaphthyl ligands and one disordered methanol molecule (molecule A), while the other has a distorted tetrahedral structure coordinated by two binaphthyl ligands (molecule B). Numerous CH···π and CH···O interactions were identified, contributing to the formation of a 3-dimensional rigid network structure. Both R -/ S -Zn exhibited fluorescence in both CH 2 Cl 2 solutions and powder samples, with the photoluminescence quantum yields (PLQYs) of powder samples being twice as large as those in solutions, indicating aggregation-induced enhanced emission (AIEE). The AIEE properties were attributed to the restraint of the molecular motion arising from the 3-dimensional intermolecular interactions. CD and CPL spectra were observed for R -/ S -Zn in both solutions and powders. The dissymmetry factors, g abs and g CPL values, were within the order of 10 ‒3 to 10 ‒4 magnitudes, comparable to those reported for chiral Zn(II) complexes in previous studies.

Supplementary files

• Supplementary information PDF (2739K)
• Crystal structure data CIF (111K)

Article information

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D. Tauchi, K. Kanno, M. Hasegawa, Y. Mazaki, K. Tsubaki, K. Sugiura, T. Shiga, S. Mori and H. Nishikawa, Dalton Trans. , 2024, Accepted Manuscript , DOI: 10.1039/D4DT00903G

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X-Ray Crystallography and It’s Applications

Jul 01, 2013

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X-Ray Crystallography and It’s Applications. By Bernard Fendler and Brad Groveman. Introduction. Present basic concepts of protein structure Discuss why x-ray crystallography is used to determine protein structure Lead through x-ray diffraction experiments

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• s1-s2 linkers
• potassium channels
• single channel analysis
• 1st experiment
• model verification
• many channels

Presentation Transcript

X-Ray CrystallographyandIt’s Applications By Bernard Fendler and Brad Groveman

Introduction • Present basic concepts of protein structure • Discuss why x-ray crystallography is used to determine protein structure • Lead through x-ray diffraction experiments • And present how to utilize experimental information to design structural models of proteins

Introduction to Protein Structure: “The Crystallographer’s Problem” • What is the crystallographer’s problem?: Structural Determination! • Structure ~ Function • Amino acids are strung together on a carbon chain backbone. • As a result: • Can be described by the dihedral angles, called φ, ψ, and ω angles. • Ramachandran Plot • Note: the crystallographer is not in the business of determining molecular composition, but determining structural orientation of a protein.

Introduction to:X-Ray Crystallography • x-rays are used to probe the protein structure: • Why are x-rays used? • λ ~ Å • Why are crystals used to do x-ray diffraction? • Crystals are used because it helps amplify the diffraction signal. • How do the x-rays probe the crystal? • x-rays interact with the electrons surrounding the molecule and “reflect”. The way they are reflected will be prescribed by the orientation of the electronic distribution. • What is really being measured? • Electron Density!!!

Performing X-Ray Crystallography Experimentsaka“Just Do It” • Bragg’s Law: • nλ =2dsin(θ) • Bragg's Law Applet • X-Ray Diffraction apparatus.

Performing X-Ray Diffraction • Resultant diffraction pattern from experimental setup • Diffraction pattern is actually a Fourier Transform of the electron distribution density.

The Fourier Transformand The Inverse Fourier Transform  

Are We Finished? • No! • 1st: We still need to determine the atomic construction (all we have is electron distribution). • 2nd: There are problems with this analysis: • The phase problem • Resolution problems • Solved with Fitting and Refinement 

Structural Basis for Partial Agonist Action at Ionotropic Glutamate Receptors • How do partial agonists produce submaximal macroscopic currents? • What is being investigated? • GluR2 ligand binding core. • Why is it being investigated? • Mechanism by which partial agonists produce submaximal responses remains to be determined. • What is going to be done? • 4 5-‘R’-willardiines will be used as partial agonists to determine the structure associated with the function. • Voltage clamping • X-ray crystallography • Outside out membrane patches for single channel analysis

Current Response • 1st experiment: • Dose Response Analysis using a two-electrode voltage clamp on an oocyte expressing the GluR2 receptor. • a.) and b.) show affinity of willardiines • Electronegativity is important • c.) and d.) show that: Size does Matter! • Note relative peak current amplitude with CTZ: • IGlu> IHW> IFW> IBrW> IIW • Note steady-state current amplitude without CTZ: • IIW > IBrW> IFW> IGlu> IHW • These data suggests that the efficacy of the XW to activate/desensitize the receptor is based on size.

Structure Meets Function • Mode of binding appears similar to glutamate • However, the uracil ring and the X produce a crucial structural change in the ligand-binding pocket.

Its all about domain closure. • Hypothesis: • the domains I and II need to be closer to produce an opening of ΔPro632 • This opening increases ion conductance.

Single Channel Analysis • They ask the question: • Do receptors populate the same set of subconductance states as with full agonists, but have different relative frequencies or open times? • To Answer the question, they first performed a fluctuation analysis of the macroscopic current by • slowly applying maximally effective concentrations of Glu, IW, and HW on outside-out membrane patches. • The weighted average conductance with Glu, HW, and IW are 13.1, 11.6, and 7.2 pS. • Suggests that the reduced efficacy reflects the activation of the open states with different average conductance.

Amplitude and Duration of Open States • To determine the amplitude and duration of the open states, a single channel analysis of the steady state responses was carried out. • Note in a and b, the distributions are the same (same conductane), so it must be that the open times of the pore for the different ligands are different.

Towards a Structural View of Gating in Potassium Channels • Ion Channel has 3 crucial elements: • Ion conduction pore • Ion gate • Voltage sensor • Architecture of Kv channels • Channel is a tetramer • N-terminus of S1 is thought to function as an intracellular blocker of the pore, which underlies fast inactivation—implies it is inside the membrane • S1-S2 linker glycosylated—outside of membrane. • S2-S3 cystein can be modified by MTS. • S3—protein toxins indicate that this is close to outside. • S4 N-terminus is accessible to MTS outside. • S4 & S4-S4 reacts to MTS inside. • S5-S6 is best defined because it remains well conserved across different channels.

Gate Structure • Pore domain is formed by S5 and S6 with S5-S6 lining the pore. • KcsA • x-ray structures support this model. • QA—pore blocker—gets stuck with rapid hyperpolarization—gate is on inside. • Further experiments indicate that the gate is on the inside. • MthK • Caught in an open state. • Pore Domains Structure and function • PVP motif (in many channels)—proline tends to kink helicies. • Increased MTS reactivity implies a larger opening with the PVP. • Metal interations not possible in the KcsA or MthK models.

Voltage Sensors: The Competing Models • S4 region is believed to be the sensor (charge rich region) • S2 & S3 have been shown to affect the voltage activation relationship. • Membrane Translocation Model • Protein charges move large distances through the membran. • Focused Field Model • Protein charges move smaller distances and focus electric field across membrane.

Model Verification!Or is it? • Note location of S4 • MT Model=yeah! • FF Model=awwh! • Some Problemos • Possible distortions in x-ray structure of KvAP • Open and closed structure mixed? • S1-S2 linkers suppose to be extracellular—from glycosylation sites experiments. • A number of other problems • Packing • MTS reactivity on both sides of membrane with approx. the same accessibility, active or not • Inconsistencies with orientations of other SX components in the structure. • Electron Microscopy shows a more expected conformation for the open position • Most noted discrepancy is that the N-terminus of S4 and S3 are probably much closer than what the x-ray structure shows.

Finally:Evidence for the Models • MTM: • Fab Fragments show biotin-avidin complexes on both sides of the membrane. Voltage sensor paddle (S3b-S4) • Red=external • Dark blue=internal • Yellow=both • FFM: • Fluorophore attatched to the N-terminal end of S4 maintains its wavelength • Energetically more favorable

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Professor Emeritus Bernhardt Wuensch, crystallographer and esteemed educator, dies at 90

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MIT Professor Emeritus Bernhardt Wuensch ’55, SM ’57, PhD ’63, a crystallographer and beloved teacher whose warmth and dedication to ensuring his students mastered the complexities of a precise science matched the analytical rigor he applied to the study of crystals, died this month in Concord, Massachusetts. He was 90.

Remembered fondly for his fastidious attention to detail and his office stuffed with potted orchids and towers of papers, Wuensch was an expert in X-ray crystallography, which involves shooting X-ray beams at crystalline materials to determine their underlying structure. He did pioneering work in solid-state ionics, investigating the movement of charged particles in solids that underpins technologies critical for batteries, fuel cells, and sensors. In education, he carried out a major overhaul of the curriculum in what is today MIT’s Department of Materials Science and Engineering (DMSE).

Despite his wide-ranging research and teaching interests, colleagues and students said, he was a perfectionist who favored quality over quantity.

“All the work he did, he wasn’t in a hurry to get a lot of stuff done,” says DMSE’s Professor Harry Tuller. “But what he did, he wanted to ensure was correct and proper, and that was characteristic of his research.”

Born in Paterson, New Jersey, in 1933, Wuensch first arrived at MIT as a first-year undergraduate in the 1950s. He earned bachelor’s and master’s degrees in physics before switching to crystallography and earning a PhD from what was then the Department of Geology (now Earth, Atmospheric and Planetary Sciences). He joined the faculty of the Department of Metallurgy in 1964 and saw its name change twice over his 46 years, retiring from DMSE in 2011.

As a professor of ceramics, Wuensch was a part of the 20th-century shift from a traditional focus on metals and mining to a broader class of materials that included polymers, ceramics, semiconductors, and biomaterials. In a 1973 letter supporting his promotion to full professor, then-department head Walter Owen credits Wuensch for contributing to “a completely new approach to the teaching of the structure of materials.”

His research led to major advancements in understanding how atomic-level structures affect magnetic and electrical properties of materials. For example, Tuller says, he was one of the first to detail how the arrangement of atoms in fast-ion conductors — materials used in batteries, fuel cells, and other devices — influences their ability to swiftly conduct ions.

Wuensch was a leading light in other areas, including diffusion, the movement of ions in materials such as liquids or gases, and neutron diffraction, aiming neutrons at materials to collect information about their atomic and magnetic structure.

Tuller, a DMSE faculty member for 49 years, tapped Wuensch’s expertise to study zinc oxide, a material used to make varistors, semiconducting components that protect circuits from high-voltage surges of electricity. Together, Tuller and Wuensch found that in such materials ions move much more rapidly along the grain boundaries — the interfaces between the crystallites that make up these polycrystalline ceramic materials.

“It’s what happens at those grain boundaries that actually limits the power that would go through your computer during a voltage surge by instead short-circuiting the current through these devices,” Tuller says. He credited the partnership with Wuensch for the knowledge. “He was instrumental in helping us confirm that we could engineer those grain boundaries by taking advantage of the very rapid diffusivity of impurity elements along those boundaries.”

In recognition of his accomplishments, Wuensch was elected a fellow of the American Ceramics Society and the Mineralogical Society of America and belonged to other professional associations, including The Electrochemical Society and Materials Research Society. In 2003 he was awarded an honorary doctorate from South Korea’s Hanyang University for his work in crystallography and diffusion-related phenomena in ceramic materials.

“A great, great teacher”

Known as “Bernie” to friends and colleagues, Wuensch was equally at home in the laboratory and the classroom. “He instilled in several generations of young scientists this ability to think deeply, be very careful about their research, and be able to stand behind it,” Tuller says.

One of those scientists is Sossina Haile ’86, PhD ’92, the Walter P. Murphy Professor of Materials Science and Engineering at Northwestern University, a researcher of solid-state ionic materials who develops new types of fuel cells, devices that convert fuel into electricity.

Her introduction to Wuensch, in the 1980s, was his class 3.13 (Symmetry Theory). Haile was at first puzzled by the subject, the study of the symmetrical properties of crystals and their effects on material properties. The arrangements of atoms and molecules in a material is crucial for predicting how materials behave in different situations — whether they will be strong enough for certain uses, for example, or can conduct electricity — but to an undergraduate it was “a little esoteric.”

“I certainly remember thinking to myself, ‘What is this good for?’” Haile says with a laugh. She would later return to MIT as a PhD student working alongside Wuensch in his laboratory with a renewed perspective.

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“He just made seemingly esoteric topics really interesting and was very astute in knowing whether or not a student understood.” Haile describes Wuensch’s articulate speech, “immaculate” handwriting, and detailed drawings of three-dimensional objects on the chalkboard. Haile notes that his sketches were so skillful that students felt disappointed when they looked at a figure they tried to copy in their notebooks.

“They couldn’t tell what it was,” Haile says. “It felt really clear during lecture, and it wasn’t clear afterwards because no one had a drawing as good as his.”

Carl Thompson, the Stavros V. Salapatas Professor in Materials Science and Engineering at DMSE, was another student of Wuensch’s who came away with a broadened outlook. In 3.13, Thompson recalls Wuensch asking students to look for symmetry outside of class, patterns in a brick wall or in subway station tiles. “He said, ‘This course will change the way you see the world,’ and it did. He was a great, great teacher.”

In a 2005 videorecorded session of 3.60 (Symmetry, Structure, and Tensor Properties of Materials), a graduate class that he taught for three decades, Wuensch writes his name on the board along with his telephone extension number, 6889, pointing out its rotational symmetry.

“You can pick it up, turn it head-over-heels by 180 degrees, and it’s mapped into coincidence with itself,” Wuensch said. “You might think I would have had to have fought for years to get it, an extension number like that, but no. It just happened to come my way.”

(The class can be watched in its entirety on MIT OpenCourseWare .)

Wuensch also had a whimsical sense of humor, which he often exercised in the margins of his students’ papers, Haile says. In a LinkedIn tribute to him, she recalled a time she sent him a research manuscript with figures that was missing Figure 5 but referred to it in the text, writing that it plotted conductivity versus temperature.

“Bernie noted that figures don’t plot; people do, and evidently Figure 5 was missing because ‘it was off plotting somewhere,’” Haile wrote.

Reflecting on Wuensch’s legacy in materials science and engineering, Haile says his knowledge of crystallography and the manual analysis and interpretation he did in his time was critical. Today, materials science students use crystallographic software that automates the algorithms and calculations.

“The current students don’t know that analysis but benefit from it because people like Bernie made sure it got into the common vernacular at the time when code was being put together,” Haile said.

A multifaceted tenure

Wuensch served DMSE and MIT in innumerable other ways, serving on departmental committees on curriculum development, graduate students, and policy, and on School of Engineering and Institute-level committees on education and foreign scholarships, among others. “He was always involved in any committee work he was asked to do,” Thompson says.

He was acting department head for six months starting in 1980, and in 1988-93 he was the director of the Center for Materials Science and Engineering, an earlier iteration of today’s Materials Research Center.

For all his contributions, there are few things Wuensch was better known for at MIT than his office in Building 13, which had shelves lined with multicolored crystal lattice models, representing the arrangements of atoms in materials, and orchids he took meticulous care of. And then there was the cityscape of papers, piled in heaps on the floor, on his desk, on pullout extensions. Thompson says walking into his office was like navigating a canyon.

“He had so many stacks of paper that he had no place to actually work at his desk, so he would put things on his lap — he would start writing on his lap,” Haile says. “I remember calling him at one point in time and talking to him, and I said, ‘Bernie, you’re writing this down on your lap, aren’t you?’ And he said, ‘In fact, yes, I am.’”

Wuensch was also known for his kindness and decency. Angelita Mireles, graduate academic administrator at DMSE, says he was a popular pick for graduate students assembling committees for their thesis area examinations, which test how prepared students are to conduct doctoral research, “because he was so nice.”

That said, he had exacting standards. “He expected near perfection from his students, and that made them a lot deeper,” Tuller says.

Outside of MIT, Wuensch enjoyed tending his garden; collecting minerals, gemstones, and rare coins; and reading spy novels. Other pastimes included fishing and clamming in Maine, splitting his own firewood, and traveling with his wife, Mary Jane.

Wuensch is survived by his wife; son Stefan Wuensch and wife Wendy Joseph; daughter Katrina Wuensch and partner Jason Staly; and grandchildren Noemi and Jack.

Friends and family are invited to a memorial service Sunday, April 28, at 1:30 p.m. at Duvall Chapel at 80 Deaconess Road in Concord, Massachusetts. Memories or condolences can be posted at obits.concordfuneral.com/bernhardt-wuensch .

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