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protein crystallisation 2
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- 1describe the set up of Xray data collection- Crystal is rotated and a diffraction pattern for each rotation is recorded
- 2what is the synchrotronThis is where high resolution data can be recorded
- This is because the Xray beam is stronger than at home source meaning that crystals diffract to a higher resolution - 3why might you choose to use the synchrotron- Lots of automation
- Takes around 2 minutes to collect data
Linear accelerator where electrons are pumped in
- Spins around the booster synxchotron where they pick up speed—> this is the big circular structure
There are many potential applications for this - 4describe the crystal rotation during crystallisation- 0.1 degree oscillation between recordings as crystal rotation
- There are between 1000-2000 images taken at around 0.1 degree oscillation per data set
- This allows for a collection of diffraction patterns - 5how are diffraction patterns of cystallography measured- The intensity of each spot is recorded and this is converted to amplitude by taking the square root
- L(hkl) is a way of measuring in 3D the 3 coordinates in an X-ray diffraction pattern
- Spacing between the crystals is analysed by the computationally; the larger the space between them the smaller the unit cell - 6what give an indication about the size of the unit suqare- Spacing between the crystals is analysed by the computationally; the larger the space between them the smaller the unit cell
- 7what is Braggs law
- 8—
- 9what does Braggs law defineBraggs law defines when there is constructive interference and when there is a large enough signal for diffraction to be measured
- Braggs law defines the condition at which the constructive interference is the strongest - 10what is the difference between 2 waves undergoing interference2dsinθ
- 11what is the wave problem and why do we need the phase informationWaves have an amplitude and a phase
- From xray experiments it is possible to measure the amplitude (from the intensity of the reflections
- However, phase angle is not measured in the diffraction pattern —> this is lost
In order to get to the point where it is possible to calculate electron density need to obtain the phase information - 12analogy for the importance of the phase informationAnalogy- If we take the amplitudes from the duck and the cat and use reverse Fourier transform to get the diffraction pattern
—> if these were combined- amplitude from the duck and the phases from the cat source and carry out reverse Fourier transform on this data then the result will equate to the information from the cat
—>this shows that the phases are the more contributing factor
—>data can be biased by the phase - 13how do we solve the phase problemThere are 3 methods used to solve phase problem
1.Isomorphous replacement (SIR) and (MIR)
2.Anomalous dispersion (or scattering)
(MAD) or(SAD)
3.Molecular replacement (MR) - 14what is isomorphous replacement- Isomorphous means the ’same as’
- Carried out by soaking the crystals in a heavy atom and also a set of crystals that does not contain the heavy atom
- The commonly used heavy atoms are mercury, platinum and lead
- It is critical that the crystals are isomorphous- meaning that the space group and unit cells are the same - 15how is isomorphic replacement data analyseddata is collected from atoms with and without the heavy atoms
--> difference gives the phase information
- 16what map is generated from the difference in the diffraction of the crystals with and without the addition of heavy atomsPatterson map
- allows the calculation of the phase information and the protein to be calculated - 17what are the limitations of isomorphous replacement- Need to find a heavy atom that binds to the protein
- Need crystals weight and without heavy atom to be isomorphous
Need more than one heavy atom - in different crystals- to remove ambiguities by combining the data - 18what is anomalous dispersionAnamalous dispersion is a change in a diffracting X-ray phase due to a strong absorbance by an atom
Anomalous dispersion results in changes the diffraction pattern- very subtle and may not be detectable by eye
Anomalous dispersion results from a heavy atom in the crystal
X-ray source needs to be close to the absorption edge of heavy atom
- Can measure single or multiple wavelengths
- Can obtain phasing information from a single crystal - 19what heavy atoms are used for anomalous dispersion- Heavy atoms do not need to be ‘as heavy’ As those needed for isomorphous replacement
- Most commonly uses selenium - 20how is selenomethionine incorporation carried out
- 21why is Selenomethionine incorporation successfulmore successful (typically) due to the protein already having the heavy metal incorporated and doesn’t require it to be soaked in metal
- 22what are the limitations of anomalous scatteringChanges in amplitudes from anomalous scattering are small so data needs to be measured carefully
- Need to be tuneable beam line at synchrotron
- Crystal can be damaged during collection due to the radiation because lots of data has to be collected - 23what is molecular replacementNeed a homologous structure available (>25% identity)
A Patterson map is calculated from amplitudes from unknown structure
- Compared to theoretical Patterson map for homologous structure
Positions of molecules are found and initial phases determined - 24schematic of molecular replacement- The known protein is taken and rotated to match the unknown and then translate and determine the values of rotation and translation To be able to be the known molecule over unknown
- Relies on overall fold of unknown molecule—> secondary fold
- Assuming the secondary fold remains the same
- 25what are the limitations of molecular replacement- Need the structure of a homologous protein
- May not work if a protein has multiple domains which are oriented differently in the model - 26how is the electron density calculated- Fourier transform is used to convert the amplitude and phase information into electron density
- 27what is the Fourier transform equation
- 28how many measurements are required to make low and high resolution density mapshigh resolution = 70 000 measurements
- side chains and folding of the proteins can be determined at this resolution
low resolution = 4000 measurements
- - Here you can see where the overall protein chain may fit but minor details cannot be visualised
- May be possible to determine the secondary structure but not able to add side chains to this structure - 29how are electron density maps calculatedComputer programmes are used to determine this data- by entering the phases and amplitude data this can trace and determine the protein backbone
- 30what is not collected experimentally by diffraction patternsphase information
- 31what information is required for the calculation of electron density maps- Electron density is calculated from amplitude and phase information
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- Protein structure and function Fichas sobre protein crystallisation 2, creado por Samantha bruce el 06/12/2019.
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- Samantha bruce
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