Molecular Replacement: Practical Application

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Molecular ReplacementPractical Application

• Richard L. Walter
• CSO, Shamrock Structures
• rwalter_at_shamrockstructures.com

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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
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
A Patterson map...looks familiar, but not quite
right
A nice search model
Let's try rotating it
We Got it!
OK...sure, you have to tumble it in a third
rotation (not shown)? ...but that's easy...so
THIS is EASY!
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The Basics of MR The Translation
t 3D translation
Correctly rotated molecule sitting at unit cell
origin
So, that looks even EASIER!
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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 Confusion
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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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Let's Get back to PRACTICAL
Our Hero
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Revisiting Rotation

• So, our model that looked so good may not be so
  good

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Revisiting Our Model

• Excellent!...but how would we know to build such
  a model a priori?

A
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...And One Other Thing
OK...we found a 2nd rotation translation
sort of ???
but there's still something wrong?
...AU contents don't have to be identical
...ANDthey have to pack reasonably!
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...But wait, that's not all!
There are LOTS of atoms in secondary structural
elements which means there are a LOT of resulting
Patterson vectors
...RIGHT or WRONG!
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What We REALLY Learned

• Happy Bunnies are Insidious Evil
• MR is Evil!
• 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 thisYou are golden
• If you do NOT see this.GIVE UP!
• Just kiddingsort of!

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Why You Often Can See This
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Why Resolution Helps

• X-ray Data between 3.5 6Å will do
• Anything higher slows you down or even hurts you

than to match these
Much easier to match these
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Its all about the starting Structure The 3D
Structure NOT the Amino
Acid Sequence
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/prfsear
  ch.html) or similar model building server.
• Many databases and servers of programs exist
  (http//mbcf.dfci.harvard.edu/cmsmbr/biotools/biot
  ools9.html\) (http//www2.uah.es/biomodel/pe/prote
  xpl/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 dont 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
Dont throw away what seems like a guaranteed MR
solution because the maps look like crap make
sure that you checked ALL enantiomorphic space
groups!
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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