Other structural methods

Description

Protein Form and Function (Methods) Mind Map on Other structural methods, created by sophie_connor on 25/05/2013.
sophie_connor
Mind Map by sophie_connor, updated more than 1 year ago
sophie_connor
Created by sophie_connor over 11 years ago
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Resource summary

Other structural methods
  1. Crystallography
    1. Understanding function: active sites
      1. Understanding similarities between proteins
        1. Understanding mechanisms
        2. Types of protein structure
          1. Not all amenable to crystallography
            1. Proteins are able to fold and are dynamic
              1. Proteins are able to form large complexes such as vesicles fusing at the neuromuscular junction
              2. Techniques for examining 3D structure
                1. X-ray crystallography
                  1. Limitation: needs a soluble proteins that will crystallise and diffract
                  2. NMR
                    1. Limitation: needs a soluble protein that is quite small
                    2. Electron microscopy
                      1. Limitation: needs a large protein that has a regular structure
                      2. Atomic force microscopy (AFM)
                        1. Limitations: in formation obtained
                        2. X-ray diffraction
                        3. Techniques for examining protein folding structure
                          1. Circular dichroism spectroscopy
                            1. Fourier transform infrared spectroscopy (FTIR)
                              1. Fluorescence
                                1. Hydrogen/deuterium exchange
                                  1. Electroparamagnetic resonance
                                  2. Assembling proteins
                                    1. Fibrous
                                      1. Extracellular matrix (elastin, collagen)
                                        1. Cytoskeleton (intermediate filaments, microtubules)
                                          1. Muscle (actin, myosin, troponin, tropomyosin)
                                            1. Amyloid (abnormally assembled protein that accumulates in disease tissue)
                                              1. Complexes
                                                1. Ribosome, proteasome, histones, clathrin
                                                  1. F1/F0 ATPase
                                                2. Circular dichroism spectoscopy
                                                  1. Differential absorption technique
                                                    1. At any given wavelength the absorption of left and right handed circularity polarised light by peptide bond is conformation specific
                                                      1. Gives rise to characteristic CD spectra
                                                      2. Measured as a function of wavelength
                                                        1. Chiral molecules (proteins) absorb left and right handed polarised light to different extents
                                                          1. Different secondary structures give different spectra
                                                            1. Advantages
                                                              1. Small amount of material needed
                                                                1. Very quick (30mins)
                                                                2. Disadvantages
                                                                  1. Difficult to interpret mixtures of structure therefore result is an average
                                                                    1. Limited information about secondary structure
                                                                      1. FTIR can be used as a complementary method to determine secondary structural elements
                                                                    2. Near UV CD
                                                                      1. Provides information about tertiary structure
                                                                        1. Detects changes in chromophores
                                                                          1. Can be used to detect folding
                                                                            1. Aromatic side chains show signals at characteristic wavelengths so can be monitored to follow changes in conformation
                                                                              1. Can be used to compare protein stability
                                                                            2. Types of spectroscopy
                                                                              1. Infrared spectroscopy
                                                                                1. Measures bond vibration frequencies in a molecule to determine functional groups
                                                                                2. Mass spectrometry
                                                                                  1. Fragments the molecule and measures the masses
                                                                                  2. Nuclear magnetic resonance
                                                                                    1. Detects signal from hydrogen atoms and can be used to distinguish isomers
                                                                                    2. Ultraviolet spectroscopy
                                                                                      1. Uses electron transitions to determine bonding patterns
                                                                                    3. Molecular vibrations
                                                                                      1. Light is absorbed when radiation frequency= frequency of vibration in the molecule
                                                                                        1. Covalent bonds vibrate only at certain allowable frequencies
                                                                                          1. Associated with types of bonds and movement of atoms
                                                                                          2. Vibrations include stretching and bending
                                                                                          3. Attenuated total reflectance
                                                                                            1. Crystal can be diamond, germanium, zinc or selenium
                                                                                              1. Signal is dependent on sample thickness and whether it is a solid or liquid
                                                                                              2. Fluorescence
                                                                                                1. Measures the environment of chromophores
                                                                                                  1. Chromophores are excited and emit at specific wavelengths and can vary depending on the environment
                                                                                                    1. Determines protein folding or compare protein folds and conformational changes
                                                                                                      1. Dye binding can be used to follow fibrillogenesis
                                                                                                      2. FTIR spectroscopy
                                                                                                        1. Similar structures can be compared using FTIR
                                                                                                          1. Advantages
                                                                                                            1. Very little material needed
                                                                                                              1. Spectra recorded very quickly
                                                                                                                1. Can be used under different conditions to monitor structural changes and differences
                                                                                                                  1. Similar to CD to allow interpretation of secondary structures compared to standard spectra
                                                                                                                  2. Disadvantages
                                                                                                                    1. Difficult to analyse and give detailed strucutural information
                                                                                                                      1. Artefacts can confuse interpretation
                                                                                                                        1. Weak spectra from some proteins
                                                                                                                      2. Atomic force microscopy
                                                                                                                        1. Measures the height of a protein above the surface in the air or liquid
                                                                                                                          1. Contact mode
                                                                                                                            1. cantilever is dragged across the surface and deflection is detected indicating damage
                                                                                                                            2. Tapping mode
                                                                                                                              1. Oscillated up and down
                                                                                                                              2. Non-contact mode
                                                                                                                                1. Oscillates above the surface (in liquid)
                                                                                                                              3. Electron microscopy for structural studies
                                                                                                                                1. Why transmission electron microscopy?
                                                                                                                                  1. Wide range of biochemical conditions
                                                                                                                                    1. Large, complex samples are suitable
                                                                                                                                      1. Can work with less than 1mg protein
                                                                                                                                        1. Crystals and high solubility not required
                                                                                                                                          1. All conformations available for analysis
                                                                                                                                          2. Transmission electron microscope images
                                                                                                                                            1. Images equivalent to those of an x-ray of a person's chest
                                                                                                                                            2. Useful for looking at structures of
                                                                                                                                              1. Macromolecular assemblies
                                                                                                                                                1. Viruses
                                                                                                                                                  1. Large particles in different states
                                                                                                                                                    1. Fibrous proteins
                                                                                                                                                      1. Membrane proteins
                                                                                                                                                      2. Carried out under a vacuum to prevent air scatter
                                                                                                                                                        1. Lenses are magnetic coils instead of glass
                                                                                                                                                          1. How is the image formed?
                                                                                                                                                            1. Electrons pass through the sample and are either...
                                                                                                                                                              1. Pass through unmodified
                                                                                                                                                                1. Scatter inelastically
                                                                                                                                                                  1. Scatter elastically
                                                                                                                                                                    1. Reflected back
                                                                                                                                                                      1. Absorbed
                                                                                                                                                                      2. Two types of contrast
                                                                                                                                                                        1. Amplitude contrast indicating loss of electrons
                                                                                                                                                                          1. Phase constant indicating interference of electrons
                                                                                                                                                                          2. Electron microscope
                                                                                                                                                                            1. An electron beam is focused onto the sample using a set of magnets
                                                                                                                                                                              1. It is then focused onto a detector screen
                                                                                                                                                                              2. Contained in a vacuum to prevent air and particle influence
                                                                                                                                                                                1. Proteins don't like being in a vacuum
                                                                                                                                                                                  1. Lenses are magnetic coils to focus the electron beam
                                                                                                                                                                                    1. EM grids are 1mm and are often made of mesh copper with a plastic or carbon layer on top to stop the protein falling through
                                                                                                                                                                                    2. Image must be processed to get an increased signal for the structure
                                                                                                                                                                                      1. Single particle averaging
                                                                                                                                                                                        1. Helical image reconstruction of fibrous proteins
                                                                                                                                                                                          1. Electron crystallography of 2D crystals
                                                                                                                                                                                        2. Negative staining
                                                                                                                                                                                          1. Specimen stained with electron dense material such as a heavy metal
                                                                                                                                                                                            1. Some of the electrons passing through the specimen are scattered by structures stained with electron dense material and appear dark
                                                                                                                                                                                              1. Others pass through parts of the specimen that are not stained to form an image on a phosphophorescent screen and appear light
                                                                                                                                                                                            2. Tells you about the morphology of the fibres but not how the chains are arranged
                                                                                                                                                                                              1. Needs more resolution
                                                                                                                                                                                          2. Cryo-TEM
                                                                                                                                                                                            1. If we want a physiologically relevant image then the sample needs to be in a physiologically relevant environment
                                                                                                                                                                                              1. Protein is in an aqueous environment
                                                                                                                                                                                                1. Protein encased in ice allows different orientations to be seen
                                                                                                                                                                                                  1. Not affected by gravity
                                                                                                                                                                                                  2. Can observe the shape of the complex and internal structures of organelles
                                                                                                                                                                                                    1. Increased resolution allows us to see subunit arrangements
                                                                                                                                                                                                      1. Able to detect conformational changes
                                                                                                                                                                                                        1. Can visualise secondary structure at high resolution
                                                                                                                                                                                                          1. Atomic resolution not possible
                                                                                                                                                                                                            1. How does it work?
                                                                                                                                                                                                              1. Ice is used to encase protein
                                                                                                                                                                                                                1. Electron microscope grid is covered in carbon and protein will find itself into one of the holes
                                                                                                                                                                                                                  1. Liquid layer will cover protein and when put into liquid ethane surrounded by liquid nitrogen it will be frozen
                                                                                                                                                                                                                    1. Ice is completely see through
                                                                                                                                                                                                                      1. You cannot see this ice through the electron microscope
                                                                                                                                                                                                                    2. Radiation damage
                                                                                                                                                                                                                      1. EM can cause damage by bombarding a protein with electrons which is very destructive
                                                                                                                                                                                                                        1. Negative stain protects from radiation damage
                                                                                                                                                                                                                        2. Cyro-TEM uses low dose of electrons to reduce damage
                                                                                                                                                                                                                          1. Damage to ice will cause melting
                                                                                                                                                                                                                            1. Results in low resolution image
                                                                                                                                                                                                                        3. Averaging noisy images
                                                                                                                                                                                                                          1. Sum noisy images together to get a clearer image
                                                                                                                                                                                                                            1. This works because signal is being added together and noise is random
                                                                                                                                                                                                                              1. Averaging many images of the same macromolecule in the same orientation can lead to increase in the signal to noise ratio
                                                                                                                                                                                                                                1. Signal is the same in all images but the noise is random
                                                                                                                                                                                                                                  1. Signal is boosted
                                                                                                                                                                                                                                2. Shared suffering
                                                                                                                                                                                                                                  1. A low electron dose can be used multiple times to produce a high resolution picture
                                                                                                                                                                                                                                    1. Full electron dose would have been lethal
                                                                                                                                                                                                                                3. Single particle analysis
                                                                                                                                                                                                                                  1. Used to look at the 3D structure of a protein
                                                                                                                                                                                                                                    1. A low image signal means you are unable to see the single molecules
                                                                                                                                                                                                                                      1. Raw images must be added together
                                                                                                                                                                                                                                        1. Orientations compared
                                                                                                                                                                                                                                          1. Reconstruct images to get a 3D image of data
                                                                                                                                                                                                                                        2. How is the information extracted from the image?
                                                                                                                                                                                                                                          1. Determine the orientation of the project with respect to the original image
                                                                                                                                                                                                                                            1. Collect sufficient projections of different orientations to give complete information on the object
                                                                                                                                                                                                                                              1. Generate 3D model
                                                                                                                                                                                                                                                1. Increase signal by averaging many projections together
                                                                                                                                                                                                                                          2. Clathrin cages in vitro
                                                                                                                                                                                                                                            1. Assemble a football like structure
                                                                                                                                                                                                                                              1. From an image you can see how clathrin fits together forming a triskeleton structure
                                                                                                                                                                                                                                            2. Icosahedral viruses
                                                                                                                                                                                                                                              1. Many viruses are arranged in shells of protein subunits with overall icosahedral symmetry
                                                                                                                                                                                                                                                1. The virus is so symmetrical that orientations can be determined using the symmetry
                                                                                                                                                                                                                                                  1. E.g. hepatitis B and Ross River
                                                                                                                                                                                                                                                2. Icosahedrons have 20 faces, 6 five fold axes, 10 three fold axes and 15 four fold axes
                                                                                                                                                                                                                                                  1. Ross River virus
                                                                                                                                                                                                                                                    1. Gives you information about the outside of the protein
                                                                                                                                                                                                                                                      1. You can peel off layers and see how the protein shell is constructed
                                                                                                                                                                                                                                                        1. Crystal structures of intracellular components
                                                                                                                                                                                                                                                          1. Greater resolution image can show secondary structures
                                                                                                                                                                                                                                                          2. Hepatitis B virus
                                                                                                                                                                                                                                                            1. Electron microscope showing virus capsids in ice
                                                                                                                                                                                                                                                              1. Narrowed down to picked out molecules
                                                                                                                                                                                                                                                                1. End up with views of the structure that become higher and higher resolution
                                                                                                                                                                                                                                                                  1. Greater resolution image can show secondary structures
                                                                                                                                                                                                                                                          3. Electron crystallography from 2D crystals
                                                                                                                                                                                                                                                            1. Some proteins will crystallise in a 2D layer
                                                                                                                                                                                                                                                              1. Electron micrograph image is collected
                                                                                                                                                                                                                                                                1. Diffraction pattern is calculated
                                                                                                                                                                                                                                                                  1. Interpretation is complicated as electrons are scattered more effectively than x-rays and may be deflected several times
                                                                                                                                                                                                                                                                2. End up with an electron density map
                                                                                                                                                                                                                                                                3. Fibrous proteins
                                                                                                                                                                                                                                                                  1. Structural proteins that provide support, shape or protection
                                                                                                                                                                                                                                                                    1. Elongated
                                                                                                                                                                                                                                                                      1. Have a specific sequence
                                                                                                                                                                                                                                                                        1. E.g. collagen, keratin or silk
                                                                                                                                                                                                                                                                          1. Electron microscopy and x-ray fibre diffraction can be used to look at them
                                                                                                                                                                                                                                                                            1. Relies on repetitive structure
                                                                                                                                                                                                                                                                            2. Do not crystallise because
                                                                                                                                                                                                                                                                              1. Insouble
                                                                                                                                                                                                                                                                                1. Heterogenous in length
                                                                                                                                                                                                                                                                                  1. Flexible
                                                                                                                                                                                                                                                                                  2. Have a crystallise structure along the fibre axis
                                                                                                                                                                                                                                                                                  3. Helical arrays
                                                                                                                                                                                                                                                                                    1. Some samples form filament or tubes with helical symmetry
                                                                                                                                                                                                                                                                                      1. Identifying the repeat or lattice allows a 3D model to be generated
                                                                                                                                                                                                                                                                                        1. All orientations of the sample are available
                                                                                                                                                                                                                                                                                          1. Examples: nicotinic acetylcholine receptor, actin, kinesin
                                                                                                                                                                                                                                                                                            1. Can calculate diffraction pattern from the tubular 2D crystal image and projection image
                                                                                                                                                                                                                                                                                              1. Diffraction pattern is an arrangment of layer lines which give information about repeating units along fibre axis
                                                                                                                                                                                                                                                                                                1. Can either be..
                                                                                                                                                                                                                                                                                                  1. Crystalline fibre
                                                                                                                                                                                                                                                                                                    1. Random orientation of crystallites around the fibre axis
                                                                                                                                                                                                                                                                                                      1. A diffraction pattern that has sharp spots on layer lines
                                                                                                                                                                                                                                                                                                      2. Non-crystalline fibre
                                                                                                                                                                                                                                                                                                        1. Polymer molecules that are not packed into crystals
                                                                                                                                                                                                                                                                                                          1. Diffraction can give continuous diffraction rather than sharp spots
                                                                                                                                                                                                                                                                                                    2. Fibres generally have helical and cylindrical symmetry
                                                                                                                                                                                                                                                                                                      1. Means they form the same way around
                                                                                                                                                                                                                                                                                                        1. Can calculate a Fourier transform
                                                                                                                                                                                                                                                                                                          1. Helix forms a series of spots
                                                                                                                                                                                                                                                                                                            1. Regular repeating pattern forms a line of dots
                                                                                                                                                                                                                                                                                                              1. A real fibre would be a combination of the 2
                                                                                                                                                                                                                                                                                                          2. Helix can be represented as a series of slits
                                                                                                                                                                                                                                                                                                            1. DNA diffraction pattern tells you about the difference between base pairs and that it is helical
                                                                                                                                                                                                                                                                                                              1. X-ray fibre diffraction and cryo-electron microscopy combined to give overall picture of fibrous protein structure and how it works
                                                                                                                                                                                                                                                                                                                1. Diffraction patterns taken when muscle is relaxed and contracted and compare
                                                                                                                                                                                                                                                                                                                  1. Crystal structures can look at different parts of the molecule
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