Structural biology Flashcards
Orders of protein structure
Primary structure: amino acid sequence from the amino (N) terminus to the carboxy (C) terminus of the polypeptide
Secondary structure: Alpha helices and beta sheets
Motifs: More organised structural element
Domain: Regions of polypeptide that folds independently
Tertiary structure: Whole polypeptide folded into a properly working protein
Quaternary structure: Multiple subunits (oligomer)
Proteins are very diverse in structure for their specific function. Can range from small soluble monomers, homo oligomers, to complex structures of membrane proteins, viral assemblies (viral capsid), microtubules etc. Some associate/assemble with nucleic acids (nucleosomes, ribosome)
X-ray crystallography methodology
Crystallisation: High-throughput crystallisation screens to find right conditions. Ex. Use Sitting drop experiment (can use automation) or Hanging drop
Assess quality. Crystals are proteins packed together with some space between and ideally have a Matthew’s coefficient of 40 (crystal has 40% water content).
Data collection: Ship liquid nitrogen frozen crystal to facility with a synchrotron. Mount protein crystal on plastic loop and beam x-rays.
Synchrotron is a particle accelerator that here accelerates electrons producing EM waves (x-rays, CD light, infrared, etc).
Monochromator is a filter that allows only X-ray to beam on protein. Beam stop is in centre of detector to stop radiation damage from primary beam of rays that weren’t diffracted by crystal
Shine (can’t focus) x-rays onto sample to obtain diffraction pattern on detector (diffraction spots recorded). Use x-rays since wavelength is at the atomic scale so allows atomic level resolution.
With Braggs law (lambda = 2dsin0 in which lambda is wavelength, d is spacing between plants in the unit cell and 0 is the reflection angle) can calculate the electron density
(Ex. famous B form DNA structure diffraction pattern taken by Roslin Franklin in 1952 at MRC biophysics unit at Kings college)
Very computer aided. As data comes in begin indexing diffraction pattern.
Rotate crystal by decided parameter (ex. one degree) then take second diffraction pattern. Too long and radiation damage occurs (burns crystal).
Structure determination: Combine the diffraction patterns, merging frames and use computation to calculate electron density map represented by mesh.
Model atomic primary structure into electron density
Validation analysis: Check Ramachandran plot for the quality of backbone torsion angles and R factors
Bioinformatics virtual ligand screening (VLS): For drug design can computationally design small molecule that would bind active site pocket to potentially inhibit.
Deposit structure in PDB. Exponential growth since the initial structure of myoglobin obtained in 1962 that won Nobel prize
Method to crystalise proteins
Crystallisation screen: In a 48 or 96 well plate each is filled with solution of crystallisation cocktail to effectively precipitate the polypeptide out (buffer, precipitates, salt or PEG, etc varying in conc. - unlimited combinations).
Adjust the cocktail to optimise (also can’t definitively predict best) and add to 24 well plate.
Add purified protein and cocktail to:
Hanging drop method: coverslip (usually 50/50) and turn 180 degrees to hang upside down on to of well on the plate or
Sitting drop method (easier to automate and perform replicates): on a pedestal in the larger well with the cocktail.
For both ensure it’s well sealed to prevent complete evaporation of water.
The protein solvent will slowly evaporate and the vapour diffuses into well and equilibrate since the cocktail in the protein solution drop is at a lower concentration than in the well.
Increase in protein and precipitant concentration causes the protein to go from soluble and stable in solution to the metastable (stable but prone to changes) heterogenous nucleation in which proteins interact more strongly in small clusters. Occurs on impurities (ex. dust) or foreign surfaces (ex. container walls) as it lowers the energy barrier for nucleation.
Supersaturation induces spontaneous homogenous nucleation (ideal in which crystals grow, forming a 3D lattice). Crystals are more pure and well-ordered than in heterogenous nucleation.
Further increases in concentration (oversaturation) causes the unstable spontaneous decomposition and protein forms aggregates or precipitates (looks like brown fluff).
Computer observes the small crystals in solution with a optical microscope and gives score. Manual inspection is necessary and verifies results
Isotropic symmetrical crystals that grew in all directions have more successful and cleaner diffraction patterns
Not plates or thin rods. Crush these and add to crystallisation solution to induce nucleation
How is the quality of a published x-ray structure judged
Resolution of data: Better than 3A to discuss ligand binding. Below 2A allows unambiguous distinction of side chains. Lower means easier to dock the sequence.
R factor: The agreement between measured and calculated intensities. Should be less than 30% to be sure the model is not overfitted and reflects data
Ramachandran plot: Phi and Psi dihedral angles should fall in allowed regions (glycine can have any). Outliers naturally occur but don’t want too many
B factor/Temperature factor: Measure of how ordered a part of the protein is (low means highly ordered, high means mobile; loops). Higher than 60 A^2 indicates a disordered structure
Facts highlighting large impact of Cryo EM
In 2017, 3 men won Nobel prize:
Bush for novel idea to freeze biological samples and look at with microscope
Frank was one of the first to develop computational method to reconstruct 3D structures from 2D projections
Henderson Developed instrumentation for microscopes and detectors
Resolution revolution in which there’s an exponential increase in CryoEM solved structures
Electron microscope inventors, how it works, resolution and example
Built by Ruska and Knoll in 1931. Won Nobel prize in 1986 for it.
In electron microscope lens is a magnetic field that focuses the electron beam (whereas light microscope uses glass). Column is a vacuum so electrons don’t lese energy and provides better beam.
Some electrons are: scattered elastically (change in direction, no change in energy or speed) or inelastically (electrons lose energy which is transferred to the sample, which causes radiation damage and noisy data) or go through the sample without any interaction, or are deflected from the original direction
Can obtain atomic resolution.
Transmission electron microscope (TEM) can record projection images just below sample. The sample is prepared on thin metal grids
CryoEM methodology
Negative stain (entry level TEM, cheap, easy sample handling at room temp.): Protein on thin carbon support film is embedded in a layer of heavy metal salts (electron dense, far more than protein) which surround the protein (light areas) like a shell. Done by adding protein sample with metal stain and blotting to remove buffer.
~15A is the best resolution that can be obtained and no internal detail is shown due to heavy signal of metal masking the protein.
Quick assay to assess different parameters in preparation (pH, salt, buffer, fractions, purification methods etc) and evaluate protein homogeneity, size, etc (confirm it’s the same size as determined by DLS)
cryoEM
Once satisfied (protein is stable, discrete particles and size/shape is similar to expected (aggregates and irregularly shaped particles can interfere with data quality). ) can perform cryoEM.
Spot sample onto gold foil grid abd blot. Plunge into liquid ethane to freeze. Can automate data collection
Diagnostic cryoEM (entry level/mid-range TEM): to evaluate protein concentration/distribution (consistent) and stability, ice thickness and uniformity, phase of ice (not crystals)
Initial cryoEM data collection (mid-range/high-end TEM): Obtain high resolution 2D classes (those in same orientation) and produce initial 3D model. Assess orientation distribution and particle yield.
High-resolution cryoEM data collection (high-end TEM): Tilt-pairs (in electron tomography image at pairs of opposite tilt angles to improve coverage), motion statistics (track movement during imaging that may affect data quality), local/overall resolution, and conformation states
Analysis:
3D model is constructed from 2D projections with:
-Single particle analysis/tomography (via angular assignment such as identifying common lines in 2D images to infer angles)
-If the protein is a larger structure, with electron tomography via (canonical tilt (ex. tilt pairs) and angular assignment)
Deposit structure in PDB and EMDB. Exponential growth since the initial structure of myoglobin obtained in 1962 that won Nobel prize
Electron tomography
Missing wedge: missing information on specific parts of the 3D shape.
Helical assembly
Some assemblies are symmetrical (essentially expanding dataset).
C2/C3/CX or helical symmetry. Have specialised software to solve to high res.
However, some are not perfectly symmetrical so working as if it’s not symmetrical may allow to obtain conformational change that’s important in biology (ex. interface between dimers).
Indicated by low res. when assuming symmetry since it’s an average of different conformations
Protein preparation for imaging
Typically used to image larger structures than in X-ray crystallography (Ribosomes)
Similar initial steps to X-ray crystallography:
Initial bioinformatics: Chose polypeptide to clone. Ex. identify long loop and only image well folded domains
Clone: Simple. Can buy DNA from company. Ex. if expression organism is bacteria should ensure codon usage is optimised for bacterial overexpression.
Overexpress: May not overexpress well so may need to choose different overexpression system (insect, mammal, etc). Bacterial is cheapest and very accessible but no PTM possible (pro for crystallisation since more homogenous sample).
Purify: Test each step of purification with SDS-Page and western blot (purity) and confirm identify (composition), as well as checking biophysics like CD, x-ray/light scattering, AUC, etc (homogeneity and stability). Perform activity assays on enzymes (Biochemical activity). Use affinity tag at C terminus for affinity or size exclusion column or test different kinds of chromatography.
Pros/Cons of EM and Crystallography
EM allows different conformational states to be obtained and can put a movie together
Don’t need to worry about if structure crystalises well (works well for those with flexible regions)
Not environmentally friendly since large datasets (lots of images taken)
Crystallography:
typically images smaller structures
//Combine both to fit the high resolution structure obtained by X-ray crystallography into an electron density map obtained via cryoEM/negative staining EM of the larger heterogenous complex in a near native state.
Fitting methods:
Manual fitting (e.g. Pymol, Chimera) to align the protein structure without altering conformation. Allows the combination of highly detailed atomic information with overall structure.
Flexible fitting (e.g. Coot, NMMD) x-ray model can deform/adjust to better fir electron density of EM map.
