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(Brief introduction to Coot/WinCoot for Model Building in X-ray Crystallography)
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| align="center"|'''This page is a template and should not be edited.'''<br><span style="font-size:90%">Click [http://openwetware.org/index.php?title={{PAGENAME}}&action=edit here], copy the source, and paste it into your page.</span>
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'''Interested in posting a protocol on OpenWetWare?  Here is a template to help you do so.''' 
'''Click the view source tab and copy everything below this line.  Paste it into your new protocol page.  Then replace the text in this page with your own protocol.  Feel free to add or delete sections as appropriate.'''
[[Category:Template]]
<!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL!  -->
==Overview==
==Overview==


Coot (Linux) is a free (for academics) model-building software used in x-ray crystallography. I found Coot to be easy to learn and more user-friendly than other model-building programs such as O or XtalView.
Replace this sentence with a brief description of the protocol and its goal.


==Materials==
==Materials==


You need to download either Coot (in Linux) or WinCoot (in Windows) and install these. Just follow the instructions on the website.
For a 50 μL PCR reaction:
[http://www.chem.gla.ac.uk/~bernhard/coot/wincoot.html WinCoot Download Site]
 
* 35 μL H<sub>2</sub>O
* 5 μL 10X PCR buffer
* 5 μL 2mM dNTPs (each)
* 1.5 μL 50mM MgCl<sub>2</sub>
* 1 μL 50μM sense primer
* 1 μL 50μM antisense primer
* 1 μL 5nM DNA template
* 0.5 μL TAQ DNA polyermerase


==Procedure==
==Procedure==
# In a PCR tube, mix the components on ice in the order they are listed above.
# Perform thermocycling program
## 95 °C 5 min
## 95 °C 30 s
## T<sub>H</sub> 30 s
## 72 °C 1 min for each 1 kb PCR product
## Repeat steps 2-4 a total of 12-36 times (24 is standard).
## 72 °C 5 min
## 12 °C hold


'''Using Skeletons in Model Building'''
==Notes==
# Open pdb file of model using File-->Open Coordinates...
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
# Open an mtz file with File -->Open MTZ, CIF or phs... This will pop up a dialogue window for map calculation. To make a 2Fo-Fc map, use the FWT and PHWT. To make an Fo-Fc map, use DELFWT and DELPHWT
#List troubleshooting tips here. 
# Now you should see both model and map in the Canvas. The 2Fo-Fc map is usually colored blue and the Fo-Fc map is colored green (positive density) and red (negative density).
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
# Click on Calculate-->Map Skeleton. In the Skeleton Dialog window, turn skeleton mode On (this will display the skeleton). You can "Color and Prune", to delete possible side chain points so your skeleton looks cleaner. This procedure creates a skeleton (points and connection) in the map. The skeleton will serve as a guide of where to build atoms.
#Anecdotal observations that might be of use to others can also be posted here.
# Put the crosshair in the Canvas to a skeleton point where a continous polypeptide chain could be observed in the map
# Open Model/Fit/Refine-->Ca Baton Mode. This builds a Ca atom at the crosshair and connects that to another point 3.8A (distance of Ca-Ca) away. You can change where the 2nd Ca is by choosing "Try Another" in the Baton window. You can also lengthen/shorten the baton with the appropriate options. When you're satisfied with the baton, click "Accept". If you make a mistake, click "Undo".
# Keep building the Ca frame until there is no clear direction on where to add Ca atom.
# In Model/Fit/Refine, choose Ca Zone-->Mainchain. Click on the range of Ca frame e.g. residue#2 and #10 that you want to have converted in mainchain atoms. Coot will process this request (takes a while, ~3min) and spits out a mainchain configuration if it can. Sometimes, the connection you make violate the geometry of mainchain atoms, in this case, nothing will be built.  


==Links==
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
#[http://www.ysbl.york.ac.uk/%7Eemsley/coot/ Paul Emsley's FAQ on Coot]
#[http://www.ysbl.york.ac.uk/%7Eemsley/coot/doc/chapters/user-manual_toc.html Coot Online Manual]


<font face="courier"><nowiki>Sehat Nauli</nowiki></font>  
==References==
'''Relevant papers and books'''
<!-- If this protocol has papers or books associated with it, list those references here.-->
<!-- See the [[OpenWetWare:Biblio]] page for more information, but this format doesn't work currently.
<biblio>
#Goldbeter-PNAS-1981 pmid=6947258
#Jacob-JMB-1961 pmid=13718526
#Ptashne-Genetic-Switch isbn=0879697164
</biblio>-->
<!-- Try the [[Template:FormatRef|FormatRef template]]-->
#{{FormatRef|Goldbeter, A and Koshland, DE|1981| |Proc Natl Acad Sci U S A 78(11)|6840-4| }} PMID 6947258
#{{FormatRef|Jacob, F and Monod, J J|1961| |Mol Biol 3(3)|318-56| }} PMID 13718526
#{{FormatRef|Ptashne, M|2004|Genetic Switch: Phage Lambda Revisited| | |Cold Spring Harbor Laboratory Press}} ISBN 0879697164


==Contact==
*Who has experience with this protocol?


==Contact==
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].
Sehat Nauli (sehat@ucla.edu)
 
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Latest revision as of 14:27, 18 June 2012

This page is a template and should not be edited.
Click here, copy the source, and paste it into your page.



Interested in posting a protocol on OpenWetWare? Here is a template to help you do so.

Click the view source tab and copy everything below this line. Paste it into your new protocol page. Then replace the text in this page with your own protocol. Feel free to add or delete sections as appropriate.

Overview

Replace this sentence with a brief description of the protocol and its goal.

Materials

For a 50 μL PCR reaction:

  • 35 μL H2O
  • 5 μL 10X PCR buffer
  • 5 μL 2mM dNTPs (each)
  • 1.5 μL 50mM MgCl2
  • 1 μL 50μM sense primer
  • 1 μL 50μM antisense primer
  • 1 μL 5nM DNA template
  • 0.5 μL TAQ DNA polyermerase

Procedure

  1. In a PCR tube, mix the components on ice in the order they are listed above.
  2. Perform thermocycling program
    1. 95 °C 5 min
    2. 95 °C 30 s
    3. TH 30 s
    4. 72 °C 1 min for each 1 kb PCR product
    5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
    6. 72 °C 5 min
    7. 12 °C hold

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

  1. Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
  2. Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
  3. Ptashne, M (2004) Genetic Switch: Phage Lambda Revisited - Cold Spring Harbor Laboratory Press ISBN 0879697164

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


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