nucleic: lec 10

Techniques like electron microscopy, x-ray diffraction, and solution scattering that rely on the way radiation interacts with matter to reveal information about objects.

Determining the distances and angles between specific groups of atoms in a molecule, providing insights into its structure.

The minimum distance between two objects that can be distinguished as separate entities in an image.

To see individual atoms as distinct objects, the imaging technique needs a resolution smaller than the distance between bonded atoms (about 1.3 Å).

The destructive effect of radiation on small molecules like proteins, often limiting the ability to obtain high-resolution images.

A type of frozen water with a glassy, amorphous structure, formed by rapid freezing to prevent ice crystal formation.

The measure of how many electrons are present in a particular area, used to create 3D images of molecules in Cryo-EM.

2D images obtained in Cryo-EM that show the molecule's shape from a specific angle, like taking a photograph of a 3D object.

A computational process to reconstruct the 3D structure from multiple 2D projection images in Cryo-EM.

The process of repeatedly improving the 3D structure in Cryo-EM by removing noisy data and aligning particles more accurately.

Cryo-EM allows imaging large, complex biomolecules without crystallization, useful for studying multiple conformations and dynamic states.

Cryo-EM is limited by the size of the molecule (requires ~200 kDa or larger), small complexes have low contrast, and it requires expensive instrumentation.

Cryo-EM offers a non-crystallization approach to study biological molecules, useful for understanding their dynamic behavior and larger structures.

The process of quickly freezing a sample in liquid nitrogen to preserve its original structure and prevent crystal formation.

The amount of electrons hitting the sample during Cryo-EM, which needs to be minimized to prevent damage and ensure high-quality images.

Cryo-electron microscopy (Cryo-EM) is a technique for visualizing and determining the 3D structure of biological molecules using a transmission electron microscope (TEM).

Samples (e.g., proteins) are rapidly frozen in liquid nitrogen to form a thin layer of 'vitreous' ice. This prevents formation of ice crystals that would distort the molecule's structure.

In Cryo-EM, electrons are transmitted through the frozen sample. Proteins are denser than water, so they scatter more electrons and appear darker, allowing us to see their shape.

Cryo-EM images are 2D projections of the 3D molecule, captured from various angles. Think of it as taking multiple photos from different viewpoints.

Thousands of projection images taken during Cryo-EM are combined, sorted, and averaged to reduce noise. Computational methods are then used to determine how the images fit together and form a 3D structure.

Cryo-EM doesn't require crystallization, making it suitable for large or flexible molecules. It can also capture multiple conformations of the same molecule.

Cryo-EM is limited by the size of the molecules that can be studied (generally needs to be ~200 kDa or larger). Small complexes have low contrast, making them harder to visualize.

The electron dose is the amount of electrons hitting the sample during Cryo-EM. Too much dose damages the sample, so it needs to be minimized to obtain high-quality images.

Iterative refinement is used to improve Cryo-EM image quality. This involves identifying and removing poor-quality data and aligning particles more precisely.

The final 3D model in Cryo-EM is an electron density map. It shows the 3D distribution of electrons in the molecule, revealing its structure.

A process where purified proteins are concentrated and mixed with precipitants (like salts) to encourage the formation of crystals. This process is essential for X-ray crystallography because the ordered arrangement of protein molecules within a crystal allows for the diffraction of X-rays.

The unique pattern of spots created when X-rays scatter off electrons in a protein crystal. The position and intensity of these spots provide information about the crystal's structure and electron density.

The challenge in X-ray crystallography where the diffraction pattern only provides the amplitude (brightness) of the scattered X-rays, but not the phase (relative position of the waves). This missing information makes it difficult to directly reconstruct the protein's 3D structure.

Techniques used to estimate the missing phase information in X-ray crystallography. Methods like molecular replacement or anomalous diffraction use existing structural information to approximate the phases.

A 3D representation of the electron distribution within a protein, created using the combined information from the diffraction pattern (amplitudes) and estimated phases. This map helps us build a model of the protein's atomic structure.

The process of creating a 3D model of the protein's structure by interpreting the electron density map. This involves placing atoms into the density map according to their positions and interactions.

A computational process that optimizes the protein model by adjusting the positions of atoms to minimize discrepancies between the calculated diffraction data and the observed diffraction pattern.

The ability to distinguish between individual atoms and their details within a protein structure. X-ray crystallography can achieve very high resolutions (around 2 Å or better).

X-ray crystallography provides a snapshot of the protein structure at one specific moment in time. It does not capture dynamic changes or flexibility of the protein over time.

NMR is a technique that exploits the magnetic properties of atomic nuclei to study the structure and dynamics of molecules.

A spin state describes the orientation of an atomic nucleus's magnetic moment, which can be aligned with or against an external magnetic field.

An external magnetic field creates an energy difference between spin states, causing more nuclei to align with the field than against it.

Precession is the wobble of an atomic nucleus's magnetic moment around the direction of the magnetic field.

Synchronized precession occurs when a group of nuclei precess in unison, creating a coherent signal.

An RF pulse is a short burst of electromagnetic radiation that can excite nuclei, causing them to transition to a higher energy spin state.

FID is the signal emitted by nuclei as they relax back to their lower energy state after being excited by an RF pulse.

Fourier transform converts the FID signal from the time domain to the frequency domain, allowing us to see the different frequencies present in the signal.

The Fourier transform (FT) is a mathematical operation that converts signals that change over time into a representation of their constituent frequencies. This means that a signal's frequency content can be analyzed.

Chemical shift refers to the change in the resonance frequency of a nucleus due to the shielding effects of surrounding electrons. It's a subtle shift that distinguishes the same type of atom in different chemical environments.

The chemical shift of a particular atom is a characteristic property. It acts as a unique 'address' for that atom within a molecule. This allows scientists to identify the specific atom within a complex spectrum.

Slight changes in the chemical shift of an atom can indicate changes in its chemical environment. These subtle shifts can be used to detect structural variations in proteins.

In protein spectra, the chemical shift of a nucleus is influenced not only by its direct chemical environment but also by the surrounding residues. Electrons from neighboring residues can shield the nucleus, affecting its resonance frequency.

Nuclei that can be detected by NMR spectroscopy, like 1H, 13C, and 15N. They have a magnetic property, like tiny magnets, allowing NMR to detect them.

Because natural proteins have low levels of NMR active nuclei (13C and 15N). To get a strong signal, we need to grow proteins in a lab with enriched 13C and 15N sources.

Each atom in a protein has a unique chemical shift, acting like an address that helps identify the atom in all NMR spectra.

They show which atoms are directly bonded to each other, like building blocks connected by specific links.

They show which atoms are close in space, regardless of whether they are bonded or not, like measuring distances between objects.

NOE data tells us which atoms are close in space. We use this information to build a 3D model that satisfies all the distance constraints.

NMR data doesn't provide a single, perfect structure but a range of plausible structures all consistent with the observed data.

They need sufficient concentration, stability, and appropriate isotopic labels. Buffers must be NMR invisible, meaning they don't interfere with the NMR signal.

NMR uses a powerful magnet to align the spins of certain atomic nuclei. By applying radio waves, it excites the nuclei and measures the time it takes for them to return to their original state, which provides structural information.

NMR relies on measuring the precession of atomic nuclei in a strong magnetic field, while scattering-based techniques, such as X-ray diffraction and electron microscopy, measure the scattering of radiation (X-rays or electrons) by molecules.

The splitting of NMR peaks due to the influence of neighboring nuclei on each other's spin.

A type of interaction between nuclei connected through covalent bonds where their spins influence each other.

A phenomenon where nuclei close in space, whether bonded or not, influence each other's spins through their magnetic fields.

A technique that uses multiple pulses and delays to collect a series of 1D NMR spectra, which are then combined to reveal complex interactions between nuclei and generate multidimensional spectra.

NMR experiments that involve two pulses with varying delays, allowing the measurement of interactions between nuclei over time and the construction of 2D spectra.

A series of NMR spectra collected at different delays.

The signal that results from the nuclei returning to their equilibrium state after being excited by an NMR pulse.

A visual representation of 2D NMR data, where the intensity of the signal is shown by the height of the contour lines.

Constraints are observations in NMR that help distinguish correct protein structures from incorrect ones.

NOEs are NMR measurements that measure interactions between atoms through space. NOEs arise from dipolar coupling, where nearby nuclei influence each other's magnetic fields.

NOEs provide distance constraints, meaning they tell us the maximum distance between two atoms. This limits the possible arrangements of the protein structure.

Coupling constants are NMR measurements that provide information about the dihedral angles between atoms. Dihedral angles describe the relative orientations of atoms within a molecule.

Chemical shifts in NMR provide information about the local environment of an atom. This can help determine if an atom is participating in a hydrogen bond or other interactions essential for protein structure, like hydrophobic packing.

NMR is strong at monitoring protein dynamics, analyzing flexible proteins, and probing local environments. It doesn't require crystals like X-ray diffraction.

NMR is challenging for large proteins (>25 kDa) and doesn't have as much automation as other methods, requiring more user interpretation.

NMR assignment aims to identify the characteristic chemical shift of each NMR-active atom in the protein. This helps scientists 'recognize' atoms in future experiments about protein structure.

A technique that uses NMR to measure interactions between two nuclei, yielding a 2D plot where peaks represent coupled nuclei.

Peaks in a 2D NMR spectrum indicate interactions between two nuclei. If two nuclei are coupled, their chemical shifts intersect at a peak in the spectrum.

A 1D NMR spectrum provides information about the properties of individual nuclei, such as their chemical shift and signal intensity.

Multidimensional NMR allows researchers to study interactions between linked atoms, revealing information about molecular structure and dynamics.

3D NMR experiments provide more detailed information about molecule structure by measuring interactions between three atoms. This helps reduce peak overlap and achieve better resolution.

NMR spectra can be used to map out the connections between nuclei in a protein, allowing researchers to identify individual peaks and their corresponding structures.

Chemical shifts provide information about the electronic environment of a nucleus. This information can be used to identify different types of atoms within a molecule.

By finding linked atoms, 2D NMR experiments identify the connections between different parts of the protein, helping to map out the full structure.