LT2 X-ray Crystallography & NMR Backbone assignment Flashcards
What type of crystal does X-ray crystallography need?
REgular, repeating array of many identical molecules (crystals) to direct an X-ray beam at
How does X-ray crystallography produce a signal?
X-ray beam onto crystal
Crystal arranged as huge number of molecules in same orientation
Scattered waves can be added up in phase and raise the signal to a measurable level
Describe crystal structure
Regular, repeating array or identical units = in each unit you may only find one protein molecule
Crystal is not a solid = contains large holes/channels filled with disordered solvent molecules
What factors need to be controlled when growing a crystal?
Can take months
Dependent on:
pH, temperature
Protein concentration
Nature of solvent and precipitant
Added ions or ligands
Gravity
By what method can you grow crystals?
Must purify the proteins first using VAPOR DIFFUSION
How do you carry out vapour diffusion?
Seal a droplet of protein solution in a chamber with a large volume of solution containing precipitant
Name two vapour diffusion methods?
Hanging drop method
Sitting drop method
Describe how the hanging drop and sitting drop method differ
The drop hangs from a siliconized glass cover over a reservoir with precipitant
Slide with depressions (seats) sealed in plastic box over a reservoir of precipitant
As the precipitant vapor diffuses into the drop, it creates a concentration gradient that encourages the protein to crystallize
Why use X-rays?
EM radiation used to visualize objects = requires the radiation to have a wavelength comparable to the smallest feature that you wish to resolve
X-ray have wavelength similar to distance between bonded C atoms ~ 1.54 angstroms
What is the function of a synchrotron?
Accelerates the electrons
What occurs when the X-rays hit the crystal?
When primary beam stroke crystal = most travel straight through it
Some are dispersed when they hit the electrons and cause the electrons to oscillate and emit X-ray in almost all direction = scattering
The X-rays emitted = interfere with each other in either destructive, constructive or anything in between manner
Define constructive vs destructive interference and how it looks when imaged
Destructive = diffracted X-rays cancel each other out
Constrictive = diffracted X-rays added together to produce diffracted beams that can be recorded as pattern
Recording can produce shades of dots anywhere in between no dot to black dot
What is the equation for Bragg’s Law?
nxwavelenth = 2d x sinθ
d = thickness of crystal
θ = incidence angle same as reflected
The condition for the two waves to stay in phase after both are reflected is that the path length CBD be a whole number (n) of wavelengths (λ), or nλ
What 3 properties define each diffracted beam?
Intensity
Phase
Position (tan2θ = r/A)
Define intensity of diffracted beam
Intensity of each diffracted spot = proportional to Amp squared (peak height) of diffracted wave
How are relative phase angles measured?
No practical way of measuring relative phase angles for the different diffracted spots
Relative phase angle = 0-360 degrees
How are electron density maps computed and what information is needed to calculate them?
Both amplitude and phase of diffracted
To compute electron density maps from diffraction pattern using inverse Fourier Transform
What does MIR stand for?
Multiple isomorphous replacement
When are heavy metal derivatives used and what for?
To obtain diffraction data of native protein crystal and at least 2 or more ISOMORPHOUS crystals
Overall phase obtained from diffraction patterns of heavy atoms
How is obtaining diffraction data from isomorphous crystal done with heavy metal derivatives?***
Usually done by diffusing different heavy metal complexes into the channels of crystal
With isomorphous crystal, the correct phase could be out of 2 possibilities
Can figure out diffraction pattern and phase with heavy metal, so only have one possibility
By comparing the two sets of data, you can determine where the heavy metal atoms are located in the crystal. These positions act as “anchors” to help you solve the phase problem.
The positions of the heavy metal atoms in the crystal are now known, and you can use this information to calculate the phases of the native diffraction data. The phases of the diffracted waves are critical for determining the actual arrangement of atoms in 3D space.
What is MAD and what does it stand for?
Multiple-wavelength anomalous dispersion
It is a powerful technique used in X-ray crystallography to determine the 3D structures of biomolecules, particularly proteins.
It exploits the anomalous scattering of X-rays by specific atoms in the crystal, typically heavy atoms or specially chosen elements like selenium.
How does anomalous scattering occur?
Heavy atom is anomalous scatterer containing protein crystal at several different wavelengths = only possible if in synchrotron
When frequency of oscillation is very similar to natural frequency of oscillation
Results in small shift in both amplitude and phase of the induced oscillation of electron
What does the strength of anomalous scattering effect depend on?
Wavelength of X-ray
Give an example of how anomalous signals are induced?
Introduce selenomethionine in place of methionine
Selenium atom replaces the S of methionine
Have strong anomalous signal a lambda that can be obtained from synchrotron
What does molecular replacement of selenomethionine in place of Met require?
HOMOLOGOUS structure
How do MIR and MAD compare?
MIR uses different heavy metals
MAD uses two type of wavelength instead
Why are electrons more effective in scattering X-rays than protons or neutrons?
Electrons have much higher charge to mass ratio than either atomic nuclei
What determines the quality of the electron density map?
Depends on resolution of diffraction data = how well-ordered the crystals are & accuracy of phases
Define the different ranges of resolution of an electron density map (low to high)
Low = more than 5A can obtain the shape of the molecule, sometimes identify alpha-helical regions as rods of electron density
Medium = 3A possible to trace path of the polypeptide chain and to fit a KNOWN a.a seq onto map
High 1.5A = one sees atoms as discrete balls of density
Does high resolution mean correct structure?
No
Need to use software to fit the side chains into the model
What is the refinement process?
Where the model is changed to minimize the difference between
- experimentally observed diffraction amplitudes
AND - those calculated for hypothetical crystal containing the model instead of the real molecule
What does the R factor express?
R factor = residual disagreement
The difference in refinement is expressed as R factor
0 = for exact agreement
0.59 = for total disagreement
Smaller R factor = more accurate structure
What is the general range of the R factor for well-determined structure?
0.15 - 0.20
What are the overall steps of X-ray crystallography?
- Protein expression, purification & crystallization
- Data collection using Imageplate
- Diffraction pattern
= inverse FT and solve phase problem of isomorphous structure - Electron density map
- Protein structure
What are the pros of X-ray cystallography?
Not limited by protein SIZE
FASTER than NMR once the phase problem is solved
What are the cons of X-ray crystallography?
Highly diffracting protein crystal needed
Not suitable for FLEXIBLE and conformational heterogenous protein
What are the prerequisites for X-ray crystallography?
Over-expression of protein
High purity, homogeneous of protein is required (needs to be rigid enough to be crystallized)
Highly soluble = 1microlitre is enough for each crystallization trial
What protein sample is needed for NMR?
Use protein in solution (don’t need to crystallize it)
NMR = strong magnet
What are two steps in NMR?***
Sequential assignment
Distance restraints (NOE)
Explain what isotopic enrichment is and why it is needed in NMR
Isotopic enrichment is the process of increasing the concentration of a specific isotope of an element in a sample
Isotopic enrichment is essential for improving NMR sensitivity and resolution, as these isotopes have non-zero nuclear spin, allowing them to produce detectable signals
Define spin angular momentum and how it is used in NMR
Certain atomic nuclei have spin values of 1/2 = SAM
In the presence of external high magnetic field, the spin angular momentum of nuclei with non-zero spin will undergo a cone-shaped rotation = PRECESSION, along the field
What does the speed of rotation depend on?
Speed depends on strength of magnetic field
Machine will have the strength in Hz = larger number, stronger field
Which is stronger, looking at H-H bonds or when we look at what is coupled to C-13 and N-15?
H-H coupling is weak = can only look at H that are 3 bonds away
Cannot look at coupling from one amino acid to another
1-bond coupling is much stronger = can look at C-C, N-C, C-H or N-H
How is NMR signal produced?
If spin value I = 1/2
Spin can only align with or against external magnetic field = low energy or high energy, respectively
Applied radio freq can induce nucleus to jump from low to high energy orienatation
When it returns to low energy state = radiation at specific frequency will be emitted (NMR signal)
Specific freq = depends on energy separation
How is NMR signal converted into a peak?
Use Fourier transformation = to go from a wave to peak
What causes a chemical shift?
The electrons because they have spin 1/2 with negative charge
The field it generates = opposes external magnetic field and provides shielding effect
With different shielding effect = effective external field is different
Thus, absorption frequency is also shifted = chemical shift
Why is chemical shift in ppm?
Chemical shift frequency change = 1 million times smaller than resonance frequency
On a 500MHz machine = 500Hz is 1ppm
What influences the frequency of the emitted radiation from each nucleus?
Depends on the molecular environment of the nucleus = different for each atom
Which atom the proton is bound
Local chemical env
Secondary structure
What are pulse sequences?
Pulse sequences are characterized by the precise timing of radio frequency pulses, delays between them, and the specific manipulation of the nuclear spins.
How are pulse sequences useful?
Allow us to look at certain interactions
By varying the timing, phase, and frequency of the RF pulses, different aspects of molecular structure, dynamics, and chemical environments can be probed
What are the differences between 1D, 2D and 3D NMR pulse sequences?
1D = a single pulse sequence in which a single RF pulse is applied, and the resulting signal is recorded.
2D = multiple pulse sequences with specific delays applied (produces COSY and NOESY)
3D = A 3D NMR spectrum provides three sets of chemical shifts, usually corresponding to three different nuclei (e.g., ^1H, ^15N, and ^13C) and the interactions between them. HNCACB
What does HSQC allow us to look at?
Only H that are attached to these N-15 labelled protein are observed
Can see backbone amide (NH) and side chain (NH2) of Asn and Gln
What problems do 2D NMR help?
Second dimension helps relieve the problem of peak overlapping
Each cross peak has 2 chemical shit values
One for H and other for N-15 of amide group
What do 3D NMR look at?
NH vs N-15 vs C-13
Can look at the amide strip of a residue
When is backbone sequential assignment used?
Identity of each peak in HSQC is unknown
Need to assign them into the known amino acid sequence
What does the 3D triple resonance experiment show?
Correlates amide H to both N-15 and C-13 using HSQC as basis
Double labeled protein is needed
In a triple resonance expt, what do different pulse sequences allow?
Allow the type of C atom that is looked at to be chosen
Either Calphai and Cbetai
Or the Calpha/beta of previous amino acid
Name the two 3D triple resonance methods
HNCACB
CBCA(CO)NH
What is the difference between HNCACB and
CBCA(CO)NH?
HNCACB gives the Ca and Cb chemical shift of the SAME residue (i) as the HN
CBCA(CO)NH gives Ca and Cb chemical shift of PRECEdiNG residue
What does the amide strip of HNCACB look like in comparison to CBCA(CO)NH?
Can see 4 signals in HNCACB = 2 for the residue and 2 WEAKER dots for the preceding residue
CBCA(CO)NH only has 2 signals showing up
What is the C-13 chemical shift value for Glycine?
Only has a Ca reading ~43ppm
No Cb
What is the C-13 chemical shift value for Alanine?
Only molecule with Cb smaller than 20ppm
What is the C-13 chemical shift value for Threonine/Serine?
Their Cb reading is larger than their Ca reading
Is it easier to assign a longer or shorter stretch of HSQC peaks?
Longer stretch = because the sequence is less ambiguous
