Molecular Replacement: “Practical” Application Richard L. Walter CSO, Shamrock Structures...
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Transcript of Molecular Replacement: “Practical” Application Richard L. Walter CSO, Shamrock Structures...
![Page 1: Molecular Replacement: “Practical” Application Richard L. Walter CSO, Shamrock Structures rwalter@shamrockstructures.com.](https://reader030.fdocuments.us/reader030/viewer/2022032801/56649d545503460f94a30670/html5/thumbnails/1.jpg)
Molecular Replacement:“Practical” Application
Richard L. WalterCSO, Shamrock Structures
![Page 2: Molecular Replacement: “Practical” Application Richard L. Walter CSO, Shamrock Structures rwalter@shamrockstructures.com.](https://reader030.fdocuments.us/reader030/viewer/2022032801/56649d545503460f94a30670/html5/thumbnails/2.jpg)
Outline & Objectives
Review the basics of MR: a technique for good or evil?
The truth of applying MR: the theoretical, the real, the ugly
Some “practical” things to try....and why you should have Mass Spectroscopist. Molecular Biologist, and Molecular Modeller friends!
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The Concept of MR
OR...Imagining Proteins to be Peanuts/Neck Pillows
}A Crystals
A Search Model
A correctly rotated Search Model A correctly
translated Search Model P …”P” is for PROTEIN
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The Basics of MR: The RotationA “Theoretical View” using “Traditional Methods”
A nice, “typical” 3 atom protein
structure
OK...sure, you have to tumble it in a third rotation (not shown)...but that's easy...so THIS is EASY!
Add Some “Patterson Vectors”
A very clear 3 atom protein Patterson...
atomic vectors added for clarity
A nice search model
A Patterson map...looks familiar, but
not quite right
Let's try rotating it We Got it!
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The Basics of MR: The Translation
So, that looks even EASIER!
t = 3D translationCorrectly rotated molecule sitting at unit cell origin
t
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So, MR is EASY...a technique for GOOD!
What could POSSIBLY go wrong!
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One Slide to TOTAL ConfusionLet's try a 5 atom protein:
...a little more confusing, but still OK...I think?
Two molecules in the cell from a dimer or just crystal symmetry)
Whoa!... That's much more complex ...and it's only 10 atoms!
What if I don't have ALL the atoms right?
So, I might get the vector positions correct...but not their magnitudes???
What if the rotation is wrong?
Some vectors STILL overlap!
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Proteins are Complex
• Average residue contains 8 “heavy” atoms
• Average protein contains 300 amino acids
• Average structure contains 2400 atoms
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OurHero
Let's Get back to “PRACTICAL”
A Protein An Asymmetric Unit A Unit Cell
A Crystal!
A “Model” for our Protein
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Revisiting RotationA Protein
22º CW
47º CW
Our “head domain” looks good...but now look at the “body-foot domain”
Our “body-foot domain” looks good...but something's not quite right about the “head domain”
• So, our model that looked so good may not be so good
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Revisiting Our ModelA Protein
• Excellent!...but how would we know to build such a model a priori?
An Improved
Model
22º CW
Now both our “body-foot” and “head” domains look good...even got some ears!
A
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...And “One” Other Thing
Our rotation & translation look
good...but the cell looks too empty
OK...we found a 2nd rotation & translation”
You have to find ALL the contents
of the AU
but there's still something wrong?
...AU contents don't have to be “identical”
…sort of ???
...AND…they have to pack reasonably!
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...But wait, that's not all!
≠ ≠ ≠because...
≠ ≠There are LOTS of atoms in secondary structural elements which means there are a LOT of resulting Patterson vectors
because...
...RIGHT or WRONG!
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What We REALLY Learned
1. Happy Bunnies are Insidious & Evil
2. MR is Evil!
3. Why would ANYONE ever do this horrible technique?
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Now that we have talked about why MR should not work…
Perhaps we can talk about how to make it work
Because….MR actually DOES work!
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The Simple Answer to “Practical”
SOLUTIONRC 1 21.96 55.01 328.44 0.0000 0.0000 0.0000 14.0 55.6 25.3 19.6 1 SOLUTIONRC 1 9.00 54.87 327.31 0.0000 0.0000 0.0000 8.0 57.1 14.2 11.0 2 SOLUTIONRC 1 39.10 75.66 28.54 0.0000 0.0000 0.0000 7.4 57.5 13.6 10.8 3 SOLUTIONRC 1 21.50 28.68 43.50 0.0000 0.0000 0.0000 7.5 57.3 15.1 10.0 4 SOLUTIONRC 1 61.63 76.42 43.19 0.0000 0.0000 0.0000 8.2 56.8 14.4 9.9 5 SOLUTIONRC 1 71.12 48.16 211.00 0.0000 0.0000 0.0000 8.4 57.0 14.4 9.8 6 SOLUTIONRC 1 59.98 50.91 330.51 0.0000 0.0000 0.0000 8.0 57.2 15.1 9.8 7
• If you see this…You are golden
• If you do NOT see this….GIVE UP!
• Just kidding…sort of!
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Why You Often Can “See This”Resolution Fold Conservation
Example 1
ALLTYROSINEKINASES
DOMAINS!
Example 2
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Why Resolution Helps
Much easier to match these
…than to match these
1.X-ray Data between 3.5 – 6Å will do
2.Anything higher slows you down or even hurts you
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It’s all about the starting Structure
The 3D Structure …NOT the Amino Acid Sequence
15% Identity 18% Identity
So how do you get a good starting structure???
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Option 1: Try a simple BLAST
• All solved structures (sure!) are deposited in the Protein Data Bank (http://www.rcsb.org/pdb/)
• BLAST your amino acid sequence (or a Swiss-Prot accession number) against the PDB structure database:
• Try http://expasy.org/tools/blast/ or http://www.ncbi.nlm.nih.gov/BLAST/
• 20%+ sequence identity usually means similar 3-D structures.
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Option 2: Structural Overlap
• Take a diverse subset of your BLAST results.
• Structurally overlap this subset using any number of available tools:
• Most graphics programs: Quanta, Coot, etc.
• On-line servers for 3D structure comparison: Combinatorial Extension (http://cl.sdsc.edu/ ), Dali (http://www.ebi.ac.uk/dali/), a good comprehensive list is at (http://en.wikipedia.org/wiki/Structural_alignment_software).
• Look for a highly conserved core and try several of the structures that closely match it or trim some of the structures down to this core.
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Option 3: Model Guided Structure ID• Submit your sequence to a threader (e.g., 3D Jigsaw:
http://www.bmm.icnet.uk/servers/3djigsaw/ ; FUGUE: http://tardis.nibio.go.jp/fugue/prfsearch.html) or similar model building server.
• Many databases and servers of programs exist: (http://mbcf.dfci.harvard.edu/cmsmbr/biotools/biotools9.html\) (http://www2.uah.es/biomodel/pe/protexpl/psbiores.htm )
• My personal favorite is the Meta server at http://bioinfo.pl/
• Throw the models themselves away but pay attention to what PDB files were used to construct the models.
• Make a list of the top 20 – 30 PDB files that were used most frequently and structurally overlap them
• Repeat “Option 2” with this test set
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Option 4: Make Friends with MS• Run your protein on an SDS-PAGE gel.
• Give the gel to a skilled Mass Spectroscopist and have her/him cut out the band, tryptic digest the extracted band, and run LS-MS.
• Have your MS friend run the tryptic fragment map against his/her database of such digests.
• Take the list of proteins IDed by the MS mapping and BLAST them against the PDB, repeating Option 1, 2, and 3 as necessary with these “hits”.
• This is a great way to quickly get the structure of a protein if you don’t even know the sequence!
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Option Last: Build Homology Models
• Take the models that were generated by “Option 2” out of the garbage and use them in MR attempts.
• Build homology models by any other standard method that you or (preferrably) a skilled modeler friend of yours uses.
• Set up heavy atoms soaks; see if you have enough sulfur anomalous single; hope that yours is an unrecognized metallo-enzyme with a reachable edge; undertake MAD.
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A Final Caveat
P41212
P43212
Don’t throw away what seems like a guaranteed MR solution because the maps look like crap: make sure that you checked ALL enantiomorphic space groups!
SOLUTIONTF1 1 21.96 55.01 328.44 0.0073 0.2403 0.2567 41.7 45.2 41.4 1 27.7 SOLUTIONTF1 1 9.00 54.87 327.31 0.8385 0.2499 0.1907 18.9 54.9 20.3 3 10.9 SOLUTIONTF1 1 39.10 75.66 28.54 0.8766 0.7239 0.1881 18.0 55.2 19.7 10 22.3 SOLUTIONTF1 1 21.50 28.68 43.50 0.8838 0.4984 0.2995 18.1 55.4 20.7 2 16.1 SOLUTIONTF1 1 61.63 76.42 43.19 0.9739 0.2338 0.3070 19.1 54.3 20.1 6 23.7 SOLUTIONTF1 1 71.12 48.16 211.00 0.7945 0.2326 0.1719 19.3 54.8 20.5 4 6.7 SOLUTIONTF1 1 59.98 50.91 330.51 0.6805 0.6670 0.0686 17.5 55.1 20.6 4 5.4
Great Solution!!
SOLUTIONTF1 1 21.96 55.01 328.44 0.0063 0.7394 0.0000 69.1 34.9 70.5 1 28.5 SOLUTIONTF1 1 9.00 54.87 327.31 0.5901 0.4253 0.4508 19.4 55.5 19.2 6 15.4 SOLUTIONTF1 1 39.10 75.66 28.54 0.1019 0.3282 0.2175 18.0 55.3 21.0 2 24.3 SOLUTIONTF1 1 21.50 28.68 43.50 0.7073 0.4348 0.4263 17.7 55.2 21.1 3 20.8 SOLUTIONTF1 1 61.63 76.42 43.19 0.2749 0.6853 0.3370 17.9 55.0 18.9 1 8.0 SOLUTIONTF1 1 71.12 48.16 211.00 0.9809 0.4550 0.0000 18.2 55.4 18.9 2 11.0 SOLUTIONTF1 1 59.98 50.91 330.51 0.3996 0.5807 0.1555 17.5 55.6 21.3 1 7.5
Even Better Solution??
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Help “In Theory” & “In Image”: A Thanks
• Artem Evdokimov – Pfizer, Inc.• Bobby Barnett – U Cincinnati• David Wishart – U Alberta• Steve Hubbard – Skirball Institute• Bart Hazes – U Alberta• Randy Read – U Cambridge• Michael Rossmann – Purdue U