IndiumIII Complexes Within the Protein Crystal After HipHop Refinement

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Indium(III) Complexes Within the Protein
Crystal After HipHop Refinement
Jan Ondrácek Institute of Molecular Genetics,
Prague
ECM23, Leuven, August 9, 2006
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HipHop Refinement
Model Generation (Hip step) reached by huge
shock in changes of solvate contents. This
enables overcoming barriers separating local
minima. Fitting to X-ray data (several Hop
steps) refinement convoluted with removal of
redundant water molecules. High number of
refinement cycles in every step.
Changes in electron density in side chain for
HIV protease in complex with inhibitor
Changes in electron density in side chain for
chain human interleukin-1b
3
Model Generation
Maxima of the difference-Fourier map at
suitable distance from the protein molecule
(between 1.4 and 3.8 Å) are defined as water
molecules. Tests revealed that the initial
number of water molecules to add should be equal
to 15 of totally determined macromolecule
atoms. The occupancies of water molecules added
are set to 0.5. Good value for their
displacement parameter is U 1.2 Å2. .
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Fitting to X-ray Data

First, at a potential water molecule position,
the initial criterion of minimum
electron-density has to be fulfilled, otherwise
the atom is excluded. The initial criterion is
gradually increased from Hop to Hop step until,
after for example five steps, a final, minimal
electron-density criterion is met.
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Second, the shape of the electron density at a
potential water molecule position is tested. At
a predefined distance (a good value from
experience is 1 Å) around the water molecule,
the electron density must be lowerthan at the
water molecule position itself. Third, the
distances of potential water molecules from
other accepted atoms, either macromolecular or
hydration shell, are tested. The minimal
distance value of 1.1 Å is used to enable us to
model proline C?disorders as a proline plus a
water molecule.
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Schema of HipHop Refinement
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Software Available

• Shelxh version well tested software. Following
• Figures and Tables are related to structures of
• porcine pancreatic elastase-Cd2 complex (1uvo),
• HIV protease in complex with inhibitor (1g35),
• amicyanin oxidized (1aac) and
• human interleukin-1 (9ilb)
• Refmac version software under testing

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Refinement Trajectories for porcine pancreatic
elastase-cd2 complex
30 HipHop cycles ------------?
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The number of refined parameters is stable for
each HipHop cycle. Their values depend only on
the resolution, the completeness and data
quality and the value of the minimal electron
density chosen. HipHop refinement can never
be over-parameterized.
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To each model of the refinement trajectory
corresponds its own unique electron
density. HipHop generates an ensemble of
electron densities.
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Electron Density Changesfor HIV protease complex
(dmin 1.80 Å)
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Multi-conformer file
for porcine pancreatic elastase-Cd2 complex
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Real Variance Of Atom Position
variance yielded by a refinement program
variance between all models
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Water Molecule Clusters
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Histogramfor porcine pancreatic elastase-Cd2
complex (1.85 Å, 0.50 e.Å-3) blue - reflections
selected for Rfree calculations red all
reflections used in refinement
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Histogramfor HIV protease in complex with
inhibitor (1.80 Å, 0.15 e.Å-3)
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Histogramfor amicyanin oxidized (1.31 Å, 0.15
e.Å-3)
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Histogramfor human interleukin-1??(2.28 Å, 0.05
e.Å-3)
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General Trends

• the ensemble of models contains additional
  structural
• information
• the use of full completeness improved R-factors
• the use of full completeness improved variances

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General Trends

• HipHop refinement improved all reliability
  values
• the improvement is better for full completeness

 
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General Trends
 

• HipHop refinement increased numbers of water
• molecules
• all values are more improved using full
  completeness

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Conclusions

• X-ray data are collected at certain resolution
• The structure is interpreted (refined,
  calculated)
• at certain electron density
• The correctness of the refinement is expressed
• by the histogram of water molecule clusters or
  
• its left/middle/right values

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Why Was Indium(III) Complexes Within the Protein
Crystal Solved?

• Indium(III) chloride is often used as a
  catalyst in
• organic syntheses.
• It was found that an organic reaction catalyzed
  by the
• mixture of indium(III) chloride and a protein
  precipitate
• is going stereospecifically.
• To determine intermediates of this reaction and
  explain its
• course, lysozyme crystals were soaked in InCl3
  solution
• and diffraction data collected to the
  resolution 1.43 Å.
• To obtain structural variances necessary for
  correct solution
• of structures of indium(III) complexes, the
  protein structure
• was refined using novel HipHop Refinement
  method.

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Final Refinement Table For Lysozyme Soaked In
InCl3 (after many previous HipHop cycles, side
chains reorientations and disorders modelling)

•  

 
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Histogram For InCl3 Soaked Lysozyme
Left/middle/right values 99/11/203
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Three Indium(III) Complexes Within InCl3 Soaked
Lysozyme Crystal
Determination of OH- and H2O In the ensemble of
models, bond lengths were averaged, variances
calculated and compared with published values
The structure of complex anion (A)
trans-dichloro-trans-dihydroxy- InCl2(OH)2(O
Leu-129)23-. The anion is symmetrical according
crystallographic twofold axis (dashed line).
coord 6
The structure of complex anion (B)
cis-dichloro-cis-dihydroxy- InCl2(OH)2(H2O)(OD2
Asp-18)2-.
coord 51
The structure of complex anion (C,D)
cis-dichloro-cis-dihydroxy- InCl2(OH)2(H2O)21-.
The anion is symmetrical according
crystallographic twofold axis (dashed line).
coord 42
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Positions Of Ions On the Surface Of Lysozyme
Molecule
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The Arrangement Around the Anion (A) for model_1,
occupancy 0.89
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The comparison of the structure of model_1 and
the structure of native lysozyme (cyan, 1iee,
0.94Å, Sauter) in the position of the anion (A)
trans-dichloro-trans-dihydroxy-InCl2(OH)2(O
Leu-129)23- 
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The Arrangement Around the Anion (B) for model_1,
occupancy 0.58
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The comparison of the structure of model_1 and
the structure of native lysozyme (cyan, 1iee,
0.94Å, Sauter) in the position of the anion (B)
cis-dichloro-cis-dihydroxy-InCl2(OH)2(H2O)(OD2
Asp-18)2-.
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The Arrangement Around the Anion (C) for model_1,
occupancy 0.32
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The Arrangement Around the Anion (D) for model_1,
occupancy 0.21
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Conclusions

• X-ray data of InCl3 solution soaked lysozyme
  crystal
• were collected at resolution 1.43 Å and
  refined at
• electron density level 0.50 e/Å3 to
  ensemble isotropic
• R 14.21 with left/middle/right values of
  probability
• histogram 99/11/203.
• Indium(III) chloride bounds selectively to
  carboxyl
• groups.
• Asymmetric indium(III) complexes in
  coordination
• 51 originating in InCl3 saturated protein
  precipitate,
• similar to complex B, can catalyse
  stereospecifically
• organic reactions.

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Related Papers

• Ondrácek, J., Weiss, M.S., Brynda, J., Fiala,
  J., Jursík, F.,
• Rezácová, P., Jenner, L.B. Sedlácek, J.
  (2005).
• Acta Crystallogr. D61, 1181-1189. (structures
  of periodates within
• a protein crystal)
• Furnham, N., Blundell, T.L., DePristo, M.A.,
  Terwilliger, T.C. (2006).
• Nature Struct. Mol. Biol. 13, 184-185. (first
  corroboration of HipHop)
• Ondrácek, J. Mesters, J.R. (2006). Acta
  Crystallogr. D,
• accepted to print. (structures of bromates
  within a protein crystal)
• Ondrácek, J., Weiss, M.S, Hilgenfeld, R,
  Sedlácek, J.
• Mesters, J.R. (2006). Structure, ready to
  submit. (general HipHop paper)
• Ondrácek,J., Streinz, L., Sieglová, I.
  Mesters, JR (2006).
• Acta Crystallogr. D, ready to submit.
  (indium(III) complexes)

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Acknowledgments

• Jindrich Haek, IMCH Praha
• Jens Nyborg, University of Aarhus
• Juraj Sedlácek, IMG Praha
• Jeroen Mesters, University of Luebeck
• Manfred Weiss, EMBL Hamburg
• Rolf Hilgenfeld, University of Luebeck

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