Determining the 3D Structures of Proteins: NMR and X-ray Crystallography

These flashcards contain key vocabulary and definitions associated with the determination of protein structures using NMR spectroscopy and X-ray crystallography.

NMR Spectroscopy

A technique used to observe local magnetic fields around atomic nuclei. The magnet aligns nuclei, looking for energy gaps between spin up and spin down. Transition between spin states gives NMR line. However, for proteins, they have lots of hydrogens, carbons, nitrogens, so they have lots of overlapping peaks.

X-ray Crystallography

A method to determine the atomic and molecular structure of a crystal by scattering X-ray beams. Take proteinsn and crystallize to semi solid state and use x-ray to determine structure. Protein crystals are very squishy. X-ray diffraction patterns are analyzed to reveal the arrangement of atoms in the crystal.

Gamma rays

Electromagnetic radiation of high energy and short wavelength.

Chemical shift (ppm)

A measure of how much the resonant frequency of a nucleus is shifted due to the electronic environment.

Proton Chemical Shift

The change in resonance frequency of hydrogen nuclei in varying chemical environments.

Nuclear Overhauser Effect Spectroscopy (NOESY)

A type of NMR spectroscopy used to identify spatially near protons in molecules. Shows us proteins that are close together in space. It provides information about proximity in the 3D structure of proteins by detecting correlations between overlapping spin systems.

Bragg’s Law

Describes the condition for constructive interference of X-rays scattered off a crystal lattice.

R-factor

A measure of the agreement between observed and calculated diffraction data.

Unit Cell

The basic repeating unit in a crystal lattice that defines the structure of the crystal.

Phase Problem

The challenge in X-ray crystallography of determining the phases of diffracted beams. The wavelength and amplitude of the diffracted beams are known, but the phases are not.

Completeness of data measurement

The percentage of measured reflections for a given resolution shell, indicating data quality.

Isomorphous Replacement

A method used in crystallography to study the effect of adding heavy atoms to crystals to aid in phase determination.

X-ray irradiation

Small monochromatic sources which use single wavelength of x-ray to check if crystal is okay, common, convenient, less expensive, but lower quality data. Big polychromatic sources, which are very intense, short exposure times works for small crystals.

Diffraction Data

Protein acts like a diffraction grafting for x-rays. Cumulative constructive and destructive interference from thousands of individual proteins. The further out a spot is from the center, the higher the resolution. The distance between spots is used to calculate the size of the unit cell.

3 ways to determine the phases

MIR, MAD, MR

MIR: Multiple isomorphous Replacement

Put heavy atoms into the crystal and see how this changes the spots by calculating a difference or “patterson” map

MAD: Multiwavelength Anomalous Diffraction

Irradiate the sample with multiple wavelengths and look for “anomalous scattering” by individual atoms

Molecular Replacement

compare to a known structure of a very similar protein, or to the same protein in a different packing environment

Electron density map

Fits the primary sequence you know to the blobs you see. We likely see nitrogen and oxygen because they are heavier. It is a three-dimensional representation of the distribution of electrons around atoms in a crystal structure, helping to visualize the arrangement of atoms.

Rfree

The R factor for a set of data not included in refinement, should be similar to R factor and definitely below 0.30

What indicated good structure

Resolution, R factor, R free, Completeness of data measurement, Precision, Heavy atoms