nucleic: lec 10
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What is the primary factor that affects the resolution of a dataset in electron microscopy?

  • The size of the detector used
  • The type of radiation used
  • The temperature of the sample
  • The distance between the observed atoms (correct)

    • What must be known for the data obtained from scattering techniques to be useful?

  • The dimensions of the sample
  • Which atoms are being observed (correct)
  • The type of radiation used
  • The properties of the contact environment

    • What is the typical resolution required to distinguish atoms as separate objects?

  • ~2.0 Å
  • ~1.3 Å (correct)
  • ~1.0 Å
  • ~0.5 Å

    • Which statement about radiation damage in small proteins is most accurate?

    <p>It takes place quickly, making macromolecules more suitable</p> Signup and view all the answers

    What is the purpose of rapidly freezing the sample in cryo-EM?

    <p>To prevent water crystallization</p> Signup and view all the answers

    Cryo-EM can image complexes smaller than 200 kDa without any challenges.

    <p>False</p> Signup and view all the answers

    What type of ice is used to embed the molecules in cryo-EM?

    <p>vitreous ice</p> Signup and view all the answers

    Match the following key features of cryo-EM with their descriptions:

    <p>Does not require crystallization = Advantage Requires specialized maintenance = Challenge Can image large biological molecules = Advantage Small complexes difficult to resolve = Challenge</p> Signup and view all the answers

    Which of the following is a challenge associated with cryo-EM?

    <p>It requires large samples for clear imaging</p> Signup and view all the answers

    Contrast in cryo-EM images is primarily generated by differences in electron density between proteins and the surrounding solvent.

    <p>True</p> Signup and view all the answers

    What technique is used to refine initial density maps in cryo-EM?

    <p>iterative improvement</p> Signup and view all the answers

    Cryo-EM employs a __________ stage in the microscope to maintain the frozen state of the sample during imaging.

    <p>cryogenic</p> Signup and view all the answers

    What is a key advantage of employing cryo-EM for imaging compared to other methods?

    <p>It can image dynamic states of particles</p> Signup and view all the answers

    What is the primary role of rapidly freezing the sample in cryo-EM?

    <p>To avoid water crystallization</p> Signup and view all the answers

    In cryo-EM, excess liquid is blotted off the sample to create a thin __________.

    <p>meniscus</p> Signup and view all the answers

    Which factor contributes to the contrast in cryo-EM imaging?

    <p>Electron density of proteins compared to the solvent</p> Signup and view all the answers

    Cryo-EM can cause significant damage to small complexes due to high electron doses.

    <p>True</p> Signup and view all the answers

    What computational methods are applied during the reconstruction of 3D structures in cryo-EM?

    <p>Back-projection</p> Signup and view all the answers

    Which of the following is a characteristic of cryo-EM's imaging process?

    <p>It generates 2D projections from multiple orientations.</p> Signup and view all the answers

    What is the purpose of using precipitants in protein crystallization?

    <p>To encourage the formation of crystals.</p> Signup and view all the answers

    X-ray crystallography can provide detailed information about protein dynamics and flexibility.

    <p>False</p> Signup and view all the answers

    Name one challenge associated with protein crystallization.

    <p>High intrinsic disorder in proteins or the need for large quantities of pure protein.</p> Signup and view all the answers

    The diffraction pattern generated in X-ray crystallography reflects the atom's __________ within the crystal.

    <p>arrangement</p> Signup and view all the answers

    Match the following terms with their definitions:

    <p>Diffraction Pattern = The resulting image from the scattering of X-rays off a crystal. Phasing = The technique used to resolve the phase problem in crystallography. Electron Density Map = A 3D representation showing the distribution of electrons in a protein. Fourier Transform = A mathematical method to combine amplitude and phase data.</p> Signup and view all the answers

    What method is commonly used to resolve the 'phase problem' in X-ray crystallography?

    <p>Molecular replacement</p> Signup and view all the answers

    Radiation damage from X-rays is a concern during data collection in X-ray crystallography.

    <p>True</p> Signup and view all the answers

    What is the typical resolution X-ray crystallography can achieve?

    <p>Around 2 Å or better.</p> Signup and view all the answers

    X-rays scatter off the __________ in the crystal to create a diffraction pattern.

    <p>electrons</p> Signup and view all the answers

    Which of the following reflects a limitation of X-ray crystallography?

    <p>It may not accommodate proteins with flexible regions well.</p> Signup and view all the answers

    What primarily determines the chemical shift of an atom?

    <p>The atom's chemical environment</p> Signup and view all the answers

    How can the chemical shift be used in spectroscopy?

    <p>As the 'address' of the atom in spectra</p> Signup and view all the answers

    What effect does a change in protein structure have on chemical shifts?

    <p>Chemical shifts may change slightly</p> Signup and view all the answers

    Which statement about the precessional frequency of atoms is correct?

    <p>Different atoms of the same element precess at slightly different frequencies</p> Signup and view all the answers

    What role do nearby residues play in the chemical shift of an atom in protein spectra?

    <p>They weakly affect the chemical shift through shielding</p> Signup and view all the answers

    What do peaks in a 2D scalar coupling NMR experiment represent?

    <p>Interactions between two coupled atoms</p> Signup and view all the answers

    Which technique is used to investigate the connections between successive residues in multidimensional NMR?

    <p>3D NMR spectroscopy</p> Signup and view all the answers

    What is the effect of increasing the dimensionality in NMR experiments?

    <p>Enhanced resolution of interactions</p> Signup and view all the answers

    What is the typical role of chemical shifts in a multidimensional NMR spectrum?

    <p>To mark the intersection of coupled atoms</p> Signup and view all the answers

    Why would one choose different frequency characteristics of the pulse in NMR?

    <p>To select specific nuclei to resonate</p> Signup and view all the answers

    What does a peak in a 1D NMR spectrum indicate?

    <p>Properties of individual nuclei</p> Signup and view all the answers

    What key information can be obtained by mapping peaks to the chemical structure of a protein?

    <p>The structural connections between nuclei</p> Signup and view all the answers

    Which amino acid characteristic can affect the chemical shift differences in NMR spectroscopy?

    <p>Presence of specific side chains</p> Signup and view all the answers

    What is the effect of applying an external magnetic field on nuclear spins?

    <p>The spins align slightly more with the field than against it.</p> Signup and view all the answers

    What happens to the energy state of the system when nuclei are excited in an external magnetic field?

    <p>The nuclei are in a higher energy state than at equilibrium.</p> Signup and view all the answers

    What is the purpose of applying a radio frequency pulse during Free Induction Decay (FID)?

    <p>To shift the sample into a non-equilibrium state.</p> Signup and view all the answers

    What characterizes the signal measured during Free Induction Decay (FID)?

    <p>It exhibits an exponential drop-off.</p> Signup and view all the answers

    How does increased magnetic field strength affect the nuclei's alignment?

    <p>It increases the energy difference, enhancing alignment.</p> Signup and view all the answers

    What is the result of synchronizing the precession of nuclei?

    <p>Nuclei precess in lockstep, creating an excited state.</p> Signup and view all the answers

    What is the primary goal of assignment in solving the NMR structure?

    <p>To identify the chemical shifts of each NMR active atom</p> Signup and view all the answers

    What key transformation is performed to convert Free Induction Decay data into frequency information?

    <p>Fourier Transform</p> Signup and view all the answers

    What is a characteristic of the equilibrium state of nuclei in an external magnetic field?

    <p>They tend to align with the magnetic field in slight excess.</p> Signup and view all the answers

    Which of the following constraints is considered the most critical in protein NMR?

    <p>NOE distance constraints</p> Signup and view all the answers

    What do NOEs measure in the context of nuclear magnetic resonance?

    <p>Interactions of atoms through space</p> Signup and view all the answers

    Which characteristic of proteins does protein NMR uniquely provide insights into compared to other techniques?

    <p>Protein dynamics and flexibility</p> Signup and view all the answers

    How do coupling constants contribute to the structural analysis in NMR?

    <p>They restrict the possible dihedral angles</p> Signup and view all the answers

    What does a hydrogen atom's chemical shift indicate in NMR analysis?

    <p>Whether it participates in a hydrogen bond</p> Signup and view all the answers

    What characteristic of Nuclear Overhauser Effects (NOEs) makes them particularly useful in NMR?

    <p>They depend on the distance between interacting nuclei.</p> Signup and view all the answers

    Why are flexible proteins or regions not a major problem in protein NMR?

    <p>NMR can effectively monitor the flexibility of regions.</p> Signup and view all the answers

    What is the main advantage of using recombinant proteins for NMR structure determination?

    <p>They can be grown with specific isotope labels.</p> Signup and view all the answers

    Which aspect of protein structure is primarily determined by through-bond coupling experiments in NMR?

    <p>The connectivity between atoms.</p> Signup and view all the answers

    Why is it necessary for buffers and salts used in NMR experiments to be NMR invisible?

    <p>They contribute unwanted signals in spectra.</p> Signup and view all the answers

    How does the chemical shift serve in NMR protein structure determination?

    <p>It specifies the atomic identity across all spectra.</p> Signup and view all the answers

    What property of atomic nuclei is essential for performing NMR spectroscopy?

    <p>Nuclear spin</p> Signup and view all the answers

    What is a potential problem that can arise during sample preparation for NMR?

    <p>Precipitation or proteolysis</p> Signup and view all the answers

    What does the ensemble of structures reported in NMR signify?

    <p>Multiple conformations consistent with data.</p> Signup and view all the answers

    How does the NMR magnet maintain the necessary conditions for spectroscopy?

    <p>Through the use of superconducting coils.</p> Signup and view all the answers

    What purpose does the isotope labeling serve in NMR structure determination?

    <p>It selectively enhances the NMR signal.</p> Signup and view all the answers

    What is the primary mechanism behind scalar coupling in NMR?

    <p>Influence through shared electrons</p> Signup and view all the answers

    How does scalar coupling affect NMR peaks?

    <p>It causes peaks to split.</p> Signup and view all the answers

    What determines the strength of scalar coupling in NMR?

    <p>Dihedral angle between atoms</p> Signup and view all the answers

    What is the purpose of varying the delay time between pulses in multidimensional NMR?

    <p>To collect spectra that differ over time</p> Signup and view all the answers

    In 2D NMR, how many pulses are typically used?

    <p>Two</p> Signup and view all the answers

    What type of plot is generated from a 2D NMR experiment?

    <p>Contour plot</p> Signup and view all the answers

    Which effect influences nuclear spins that are not directly bonded in NMR?

    <p>Nuclear Overhauser Effect</p> Signup and view all the answers

    Which condition is necessary for scalar coupling to occur?

    <p>Atoms must be connected by at most three bonds</p> Signup and view all the answers

    Study Notes

    Microscopy and Scattering Techniques

    • Electron microscopy, X-ray diffraction, solution scattering, and cryo-electron microscopy (cryo-EM) use scattering to analyze samples.
    • These methods measure distances and angles between atoms to understand atomic arrangement, but require knowledge of which atoms are being observed.
    • NMR, SPR, FRET, and cryo-EM are similar scattering techniques.
    • Analyzing scattered radiation's properties (direction and phase) allows researchers to create images or models of the scattering object.

    Resolution and Limits

    • Resolution is the smallest separation at which objects can still be distinguished.
    • Objects closer than the resolution are combined into one observation peak.
    • To distinguish individual atoms, a resolution better than their bonded distance (~1.3 Å) is required.

    Radiation Damage and Macromolecules

    • Radiation damage occurs quickly for small proteins, thus macromolecules are used to avoid this.
    • Cryo-EM uses very low electron doses to minimize damage.
    • High-energy X-rays can damage crystals during X-ray diffraction data collection.

    Cryo-Electron Microscopy (Cryo-EM)

    • Sample Preparation: Proteins or other biological molecules are applied to a carbon grid. Excess liquid is blotted off, leaving a thin meniscus, and the sample is rapidly frozen in liquid nitrogen to form vitreous ice, embedding the molecules.
    • Imaging: A cryogenic stage maintains the sample's frozen state in the transmission electron microscope (TEM). Electrons are transmitted through the sample to produce images. Contrast is generated because proteins are more electron-dense than the surrounding solvent. Many images are necessary for structural determination due to the low contrast.
    • Data Collection: Cryo-EM images are 2D projections of the sample. Images from different viewing angles are collected to prevent damage and maximize data, using limited electron doses to prevent damage, yielding low contrast, necessitating many images.
    • Image Processing: Thousands of images from a variety of orientations are processed computationally. Images are aligned and averaged to reduce noise and artifacts. The relative orientations of the images are determined. This step is often computationally intensive.
    • 3D Structure Reconstruction: Projections from the numerous images are computationally back-projected to create a 3D electron density map.
    • Structure Refinement: Initial density maps are refined iteratively. Poor images are discarded, and particles are aligned more precisely, capturing various views to improve resolution. Rare views not initially included can be added during refinement. Software and computational power are critical for refinement.

    X-ray Crystallography

    • Protein Crystallization: Purified protein is concentrated and mixed with precipitants (e.g., salts, organic polymers) under controlled conditions to encourage crystal formation. Specific interactions between protein molecules in the crystal ensure long-range order and translational symmetry in three dimensions. Requires large quantities of pure protein (~10 mg or more). Proteins with high intrinsic disorder or flexible regions may fail to crystallize.
    • Data Collection: The crystal is exposed to intense, monochromatic X-ray beams. X-rays scatter off electrons in the crystal, creating a diffraction pattern recorded by a detector. The pattern consists of spots, whose positions and intensities carry information about the crystal's unit cell and electron density.
    • Diffraction and Interference: X-rays scattered by multiple atoms interfere constructively and destructively. This interference generates a unique diffraction pattern for the crystal, which reflects the atomic arrangement.
    • Phasing and Electron Density Maps: The diffraction pattern provides amplitude data but lacks phase information (the "phase problem"). Phasing methods like molecular replacement or anomalous diffraction are used to approximate the phases; a Fourier transform combines amplitude and phase data to construct a 3D electron density map of the protein.
    • Model Building and Refinement: The electron density map is interpreted to build a model of the protein's atomic structure. Computational refinement optimizes the model by minimizing discrepancies between observed and calculated diffraction data. The final structure is evaluated for quality and precision.

    Key Features of Cryo-EM

    • Advantages: Cryo-EM doesn't require crystallization, is suitable for large and complex molecules, and can capture multiple conformations and dynamic states.
    • Challenges: Clear imaging requires samples larger than ~200 kDa. Small complexes have low contrast, making resolution challenging. Cryo-EM instrumentation is expensive (approximately $10 million) and requires specialized maintenance; extensive data processing is necessary for reliable structures.

    Key Features of X-ray Crystallography

    • Advantages: Typically achieves resolutions around 2 Å or better; works for proteins of all sizes if well-folded; captures the protein, bound water, ions, ligands, and covalent modifications.
    • Challenges: Requires large quantities of pure protein; proteins with high intrinsic disorder or flexible regions may fail to crystallize; provides a snapshot of the structure but no information on dynamics or conformational flexibility; phases must be inferred indirectly, which adds complexity; sophisticated crystal growth and diffraction techniques are necessary.

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    Protein Structure Determination using NMR PDF

    Description

    Test your knowledge on various microscopy and scattering techniques such as electron microscopy, X-ray diffraction, and solution scattering. Explore the concepts of resolution, radiation damage, and how these methods are utilized in analyzing atomic arrangements. This quiz will assess your understanding of these advanced scientific principles.

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