Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011

 
Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
Challenges For Structure Determination
          At Low Resolution

               Axel Brunger
            Stanford University

PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
Letter

                                    PUBLIC ACCESS TO X-RAY D I F F R A C T I O N D A T A

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 the preservation of important and expensively determined data which otherwise                                                                     are likelyA venue
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 lost with the ever-changing personnel and computer installations in numerous                                                        New Haven,         laboratories
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 ing Agarwal,
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Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
The Low Resolution Problem

       How can one obtain a “good” crystal
          structure at low resolution?

There are generally more independent reflections than
           torsion angles at 5 Å resolution

  Problems:
   • poor observable to parameter ratio
   • electron density maps may be difficult to interpret
   • potential model bias

  Need for powerful reciprocal and real space methods
Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
Outline

•   Overview of DEN-refinement

•   MR for a new structure at 3 Å (high B-factors)

•   Refinement at 7.4 Å resolution
Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
Outline

•   Overview of DEN-refinement

•   MR for a new structure at 3 Å (high B-factors)

•   Refinement at 7.4 Å resolution
Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
Target Function For Macromolecular Refinement

                                      macromolecular structure
                                      knowledge

    Etotal = Egeometric + wx-ray Ex-ray + wmolecule Emolecule

improved
      chemical → improved phases → improved
         model knowledge                     electrondata
                                                      density maps
                                         diffraction
Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
in order to achieve a good fit to the data. If the cutoff
       isDEN (Deformable
          added          Elastic Network)
                from the reference model.Restraints
                                          The reference mo
                      18 13
       modeling               and should have good local geometry

           Etotal = Egeometric + wx-ray Ex-ray + wDEN EDEN
       The elastic network energy term comprises a sum ov
                                                              4p
                   E DEN (n) =        ! (d        ij   − d (n))
                                                         0
                                                         ij
                                   N pairs i, j

      where
Reference            is theinter-atom
          modeld➔ij selected distance     between
                                      distances      atom
                                                ➔sparse       pairs
                                                        distance     i and
                                                                 network d0ij(n)j   i
 • randomly selected atom pairs within a specified distance range,
     the corresponding          equilibrium distance at              DEN upda
     and separated by a specified interval in the primary sequence
 •   typically ~ 1 distance restraint per atom
       corresponding distance of the  reference model. The e
                                   Schröder, Levitt, Brunger, Nature 2010
Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
in order to achieve a good fit to the data
                Implementation Of The  DENfrom
                                   is added Method
                                               the reference model. The
Several (typically 10) macrocycles consisting of:                                18 13
                                                                 modeling                and should have good lo
  • torsion angle refinement        (slow-cooling                  molecular
                           Chapter 3. Principles of Protein Structure.
                                                                                 dynamics)Use restricted to students enrolled in Chem130/MCB100A
  • default option: restrained grouped B-factor refinement                                                                     UC Berkeley, Fall 200

  • default option: last two cycles
                                The peptide    aregroupwithout shown DEN      restraints
                                                                       in Figure 3.4A is said to
Target function:                                                       The elastic network energy term compr
                        be in the trans conformation because the two C! atoms
                        are on opposite sides of the peptide bond (the C! atoms
                                                                                                              4p
      E
     total      =E
             geometric  C! aatoms
                                     +w E
                                  MLare on theDENsameDEN
                                                         +w
                        are said to be staggered). The alternative, in which the
                                                                      E
                                                         side of theDEN
                                                                     peptide bond
                                                                                      E     ij  (n) =0
                                                                                                     ij       ! (d           − d (n))
                                                                                  FIGURE
                                                                                      i, j  PRO?STRUCT??V
                        (Figure 3.4C), is referred to as the cis conformation,N pairs
During slow-cooling cycles,
                        and periodic
                             is rarely seenupdates
                                             because itof
                                                        brings the R groups, some
the DEN equilibrium distances
                        of which are    performed
                                   are quite bulky, into close contact.
                                                                  ij   where d is the distance between atom
                                                                                                 Y

                                                                                                                        F
                                         3.2                           the corresponding equilibrium
                                            The backbone torsion angles # (phi) and $                distance
                                            (psi) determine the conformation of the protein    Y
   γ: deformation factor             (adjustable)
   wDEN: weight for DEN restraints
                                            chain
                                          (adjustable) corresponding distance of the reference
                                  Because of the planarity of the peptide groups, a         F
   κ: damping factor (usually     setbackbone
                             protein   to 0.1)only has freedom to rotate at the                Y
   drefij: distances from reference    model
                             points where              updated      every     six
                                            the peptide groups meet (Figure 3.5).  torsion angle  molecu
                             These swivel
   doij(n): current DEN restraint          points are at each C! atom of the peptide
                                      distances        by by single bonds to the            F
   dij: current distances ofchain.   The C! atom is connected
                              refinement      model
                             carbonyl carbon (C) and the amide nitrogen (N) of the
Multiple trials for each (γ,same residue. Rotation about the N–C! and C!–C
                               wDEN) parameter pair and temperatureij                 d 0 (n + 1) = (1− κ )dij0 (n) + κ [γ dij
                            bonds define two torsion angles, denoted # and $,
Trial with the best Rfree isrespectively.
                              used for By subsequent     steps
                                            convention, positive rotation in # and
                                                                       where the parameter κ specifies the spe
Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
How Does It Work?
• DEN is a general refinement method that guides torsion angle
  molecular dynamics by restricting it to reasonable conformational
  changes (e.g., differences between homologous models or motions)
• DEN introduces information that is specific for the particular starting
  structure (e.g., homology model)
• Degree of deformation is determined by γ
 • γ=0: no deformations of reference distances allowed
 • γ=1: deformations track refined model (akin “jelly body”)
 • 0
Challenges For Structure Determination At Low Resolution Axel Brunger Stanford University - PDB40 Symposium, Cold Spring Harbor Laboratory, 2011
Some Notes And Considerations

• General modes
  • New refinement: initial model = reference model
 • Re-refinement: initial model ≠ reference model
• Special options
  • DEN-restraints active throughout
 • wDEN=0 ↔	 torsion angle simulated annealing (without DEN)
• Implementation in CNS 1.3 and Phenix.refine (development)
• Resource for grid search: SBGrid Science Portal (www.sbgrid.org)
Outline

•   Overview of DEN-refinement

•   MR for a new structure at 3 Å (high B-factors)

•   Refinement at 7.4 Å resolution
Structure Of Cgl1109 (JCSG HP3342)
    (A Putative Succinyl-Diaminopimelate Desuccinylase
                (dapE) From C. Glutamicum)
            Collaboration with D. Das, A.Deacon, J.Grant,
       T. Terwilliger, R. Read, P. Adams, M. Levitt, G. Schröder

•   Anisotropic diffraction data to ~ 3 Å resolution

•   MAD data available, but experimental map was not easily
    interpretable

•   Weak homology to known structures (1vgy with 25% identity)

•   One of the cases used by DiMaio et al. (2011)

•   MR solution for both 1vgy and Modeller model of Cgl1109
    (Phaser: RFZ=3.2, TFZ=9.9, LLG=75, Rcryst=0.65)
DEN Refinement
    + Automated Model Building With AutoBuild

•    Reference model = initial model = MR solution
     (search model: Modeller homology model)

•    Standard DEN protocol: but restrained individual
     B-factor refinement

•    AutoBuild with “morphing” and “rebuild in place”
Optimization Of DEN Parameters
        optimum
        Rfree

        100.0
  DEN wDEN

                                                      Rfree
             10.0
Weight

                                                      0.475

                                                      0.470
w

                                                      0.465

              1.0                                     0.460

                                                      0.455

                                                      0.450

                                                      0.445
              0.0
                    0.0   0.2   0.4       0.6   0.8           1.0
                                 Parameter γ
                                      γ
Larger Radius Of Convergence For DEN Vs. Standard Refinement
Standard refinement (Rfree=0.52) vs. final (orange)   Standard ref. + AutoBuild (Rfree=0.48) vs. final (orange)

       DEN (Rfree=0.44) vs. final (orange)             DEN + AutoBuild (Rfree=0.42) vs. final (orange)
DEN-Refinement Produced Better 2mFo-DFc Maps
                  Than Standard Refinement

Standard ref. (blue) + AutoBuild (cyan)           DEN (blue) + AutoBuild (cyan)

                            Orange: final model
DEN+Autobuild Map Showed How To Correct The Model

      Magenta: model after first round of DEN+AutoBuild
      Cyan: corresponding DEN+AutoBuild 2mFo-DFc map
      Orange: final model
Final Model
        Rfree                       0.258
       Rcryst                       0.234
Ramachandran favored                92.7%
Ramachandran outliers               0.8%
  Molprobity score          2.41 (96th percentile)
                  Final 2mFo-DFc
Results For The Refinement Of Cgl1109

• Synergism between DEN-refinement and AutoBuild
• Improved model ➔ improved phases ➔ better starting point
  for AutoBuild
• Semi-automated completion of the structure with AutoBuild
Outline

•   Overview of DEN-refinement

•   MR for a new structure at 3 Å (high B-factors)

•   Refinement at 7.4 Å resolution
Refinement Of Photosystem 1 (PSI) At 7.4 Å Resolution
        Collaboration with Henry Chapman,Thomas White (CFELS, DESY),
               Petra Fromme, Raimund Fromme (Univ. of Arizona)

•   DEN-refinements using synchrotron (ALS) data of a PS1 crystal in harvesting
    buffer used for nano-crystal generation (Chapman et al., Nature, 2011),
    dmin ~ 5 Å

•   Data truncated to 7.4 Å to make it comparable to FEL (LCLS) data of PS1

•   Increasingly scrambled starting models (Cα rmsd to target 1JB0: 0, 2.3,...,4.4 Å)

•   Omitted three Fe-S clusters (for validation)

•   Reference model = starting model

•   Standard DEN protocol, except DEN active throughout, sparse random selection
    between all atoms, and only overall B-factor refinement
Increasingly Scrambled Starting Structures

 Green: PS1 (PDB ID1JB0)
 Magenta: maximum scrambled structure
 (Cα rmsd: 4.4 Å, right most helix displaced by 5.4 Å)
Molecular Replacement (7.4 Å Resolution)

                     Phaser TFZ Score                                 Phaser LLG Score
      30.0                                              400

      22.5                                              300

                                                  LLG
TFZ

      15.0                                              200

       7.5                                              100

        0                                                0
             1   2       3       4        5   6               1   2       3       4        5   6
                     Starting Structure                               Starting Structure

                          Solutions were found for all starting structures
Comparison Of Refinement Protocols

                          Rfree                                    RMSD To Target
        0.55                                             7

        0.50                                             6

                                              RMSD (Å)
                                                         5
        0.45
Rfree

                                                         4
        0.40
                                                         3
        0.35
                                                         2

        0.30                                             1

        0.25                                             0
               1     2    3   4    5    6                     1    2    3   4    5     6

                   Starting structure
                                            Average Significance () of FeS Peaks
                                                         12

overall rigid body refinement                            9

standard refinement
                                             
                                                         6
torsion simulated annealing refinement
DEN-refinement                                           3

secondary structure restrained refinement
                                                         0
                                                              1    2    3    4   5     6

                                                                  Starting structure
Results For Maximally Scrambled Starting Structure

                                                   Mg ions are shown as spheres

DEN (orange) vs. starting (magenta)    DEN (orange) vs. target 1JB0 (green)
                   DEN-refinement shifted helices by 5.5 Å
Results For The Maximally Scrambled Starting Structure

                                                        Mg ions are shown as spheres

  DEN 2mFo-DFc map (blue)                          target 2mFo-DFc map (blue)
   vs. target 1JB0 (green)                            vs. target 1JB0 (green)
                             1.5 σ contour level
DEN-Refinements With Two Helices Omitted
            (Chain F, Residues 103:126 - Yellow)
DEN-refinement starting from 1JB0                     DEN-refinement starting from max. scrambled

    DEN mFo-DFc map (mesh)                                   DEN mFo-DFc map (mesh)
vs. target 1JB0 (green and yellow)                       vs. target 1JB0 (green and yellow)

                     3 (orange), 2.5 (blue), 2 σ (light blue) contour levels
Results For The Maximally Scrambled Starting Structure:
                 Standard Refinement
                  Mg ions are shown as spheres

                  standard refinement (teal)
                    vs. target 1JB0 (green)
Results For Maximally Scrambled Starting Structure:
              Standard Refinement
               Mg ions are shown as spheres

           standard refinement 2mFo-DFc map (blue)
                    vs. target 1JB0 (green)
                    1.5 σ contour level
DEN-Refinement At 7.4 Å Resolution Is Beneficial

  Compared to overall rigid body, standard, or simulated
  annealing refinement, DEN-refinement

    • moves closer to the true structure
    • recovers information not included in the refined
      model - more significant features in difference
      maps
Overall Conclusions For DEN-Refinement

• DEN-refinement is a general method that can produce
  better models than standard refinement or simulated
  annealing protocols
• DEN-refined model phases are a better starting point
  for (automated) model building
• Most benefit is for starting structures that are far from
  the true structure, low-resolution, or high B-factor cases
Acknowledgments
DEN refinement
  Gunnar Schröder, Michael Levitt

Cgl1109/HP3342 structure determination
  Debanu Das, Tom Terwilliger, Randy Read,
     Paul Adams, et al.

DEN-refinement of photosystem 1
  Henry Chapman, Thomas White,
    Petra Fromme, Raimund Fromme, et al.

SBGrid Science Portal
  Dan O’Donovan, Piotr Sliz
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