Overview how to get the 3D protein structure and phasing issue of X-ray
A brief overview of how to get the 3D protein structure.
1. gene of interest2. cloning it 3. expressing it4. purify it5. crystallize it - not really depend on protein size, it is the matter of luck, patient, and good observations.
Three methods; microbatch -- good for screening (sample and precipitant are mixed and covered by the paraffin oil), vapor diffusion -- hanging drop (using the exchange between precipitant and reservoir), liquid diffusion -- hard to implement
To solve with Synchrotron - the crystal should be in the range between 20-200 micrometers.
Way to differentiate the protein crystal from the salt; staining with Coomassie after running the SDS-PAGE or directly checking from the diffraction - salt and protein give a very different diffraction pattern.
6. X-ray diffraction--phase--electron density map obtain--fitting--building the model!7. Analyze data8. Submit the 3D information at PDB
Protein crystallography really depends on luck and it is the fine piece of art. The speaker mentions that the ribosome structure that we have seen in the textbook to this day, it has taken 20 years much of the afford and passing the study through generation by generation.
I have just realized that to get the correct data from X-ray diffraction, the technique to get the correct phase (my understanding is it would help the mapping between electron density and AA sequence more realistic) is very important. There are four methods that the speakers mentioned in the lecture.1. molecular replacement where you can get the template from the deposited PDB. However, it is possible that it might not be correct due to the homology between the query and subject. Therefore, you might get the less precise structure.2. Multiple/single isomorphous replacement//for one wavelength can use in-house but for multi-wavelength must require higher facility like synchrotron.//getting phase info from one native and another dipping in heavy atom solution. However, it requires the same unit cell and same position of mlc in the unit cell.
There are more details on this method -- by dipping in the heavy atom solution - it may interfere with the unit cell as well as the position in the unit cell. Besides, it requires very good interaction between the protein and heavy atoms.
3.Multiple/single wavelength anomalous dispersion//requires the synchrotron facility -- the protein must be either metalloprotein or selenomethionine proteins (as I discussed on this issue - just realized that the media to replace S with Se is very expensive and if the Met is at the most end in any either side - it is not a good candidate for this method). The suitable protein should have met at least 3, so the phase could be calculated from the data. Heavy atom derivatives can be used with this technique.
4.Direct method - explain very little due to this technique is rarely used and it can be only applied for a small molecule.
There are two programs that were introduced and used in this workshop;
1. CCP4 -- to analyze the data from the diffraction
http://www.ccp4.ac.uk/
2. PyMol - This one uses for 3D visualization - requires the license key but it can be used for free within 30 days.
https://pymol.org/2/
1. gene of interest2. cloning it 3. expressing it4. purify it5. crystallize it - not really depend on protein size, it is the matter of luck, patient, and good observations.
Three methods; microbatch -- good for screening (sample and precipitant are mixed and covered by the paraffin oil), vapor diffusion -- hanging drop (using the exchange between precipitant and reservoir), liquid diffusion -- hard to implement
To solve with Synchrotron - the crystal should be in the range between 20-200 micrometers.
Way to differentiate the protein crystal from the salt; staining with Coomassie after running the SDS-PAGE or directly checking from the diffraction - salt and protein give a very different diffraction pattern.
6. X-ray diffraction--phase--electron density map obtain--fitting--building the model!7. Analyze data8. Submit the 3D information at PDB
Protein crystallography really depends on luck and it is the fine piece of art. The speaker mentions that the ribosome structure that we have seen in the textbook to this day, it has taken 20 years much of the afford and passing the study through generation by generation.
I have just realized that to get the correct data from X-ray diffraction, the technique to get the correct phase (my understanding is it would help the mapping between electron density and AA sequence more realistic) is very important. There are four methods that the speakers mentioned in the lecture.1. molecular replacement where you can get the template from the deposited PDB. However, it is possible that it might not be correct due to the homology between the query and subject. Therefore, you might get the less precise structure.2. Multiple/single isomorphous replacement//for one wavelength can use in-house but for multi-wavelength must require higher facility like synchrotron.//getting phase info from one native and another dipping in heavy atom solution. However, it requires the same unit cell and same position of mlc in the unit cell.
There are more details on this method -- by dipping in the heavy atom solution - it may interfere with the unit cell as well as the position in the unit cell. Besides, it requires very good interaction between the protein and heavy atoms.
3.Multiple/single wavelength anomalous dispersion//requires the synchrotron facility -- the protein must be either metalloprotein or selenomethionine proteins (as I discussed on this issue - just realized that the media to replace S with Se is very expensive and if the Met is at the most end in any either side - it is not a good candidate for this method). The suitable protein should have met at least 3, so the phase could be calculated from the data. Heavy atom derivatives can be used with this technique.
4.Direct method - explain very little due to this technique is rarely used and it can be only applied for a small molecule.
There are two programs that were introduced and used in this workshop;
1. CCP4 -- to analyze the data from the diffraction
http://www.ccp4.ac.uk/
2. PyMol - This one uses for 3D visualization - requires the license key but it can be used for free within 30 days.
https://pymol.org/2/
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