A Primer in X-ray Crystallography for Redox Biologists. Mark Wilson Karolinska Institute June 3 rd, 2014

Transcription

1 A Primer in X-ray Crystallography for Redox Biologists Mark Wilson Karolinska Institute June 3 rd, 2014

2 X-ray Crystallography Basics Optimistic workflow for crystallography Experiment Schematic Fourier Transform -1 Fourier Transform Monochromatic X-rays typically used

3 Common Crystallographic Terminology Resolution: Usually measured in Ångstroms; higher resolution corresponds to smaller number Unit cell: Basic building block of crystal; can generate the entire crystal by translation (straight-line motion) Asymmetric unit: Most fundamental unit of crystal; must be rotated/translated to make the unit cell Space group: collection of symmetry operations that build the unit cell from the asymmetric unit Phase: A quantity that is required to calculate electron density maps but cannot be directly observed in the crystallographic experiment R/R free : A measure of model quality; fractional disagreement between model and data

4 The Unit Cell, Asymmetric Unit, and Space Group The unit cell contains a number of molecules related by certain symmetry operators The most fundamental part of the crystal is the irreducible structural elementasymmetric unit The collection of symmetry operations that generate the unit cell from the asymmetric unit compose the space group Symmetry operation here is the two-fold bioassembly_tutorial.html

5 Why Crystallographers Worry About Phases The amplitudes, but not phases, of the structure factors are experimentally measured Duck F Cat φ Fourier transform Fourier transform -1 Cat F Duck φ Problem: The phases, not the amplitudes, completely dominate the inverse transform The data we need most are the data we cannot directly measure

6 Structures Are Models of Electron Density Calculate density Build model Diffraction data (one of ~360 images) Electron density with model

7 R = Quantifying Model Quality: The R Value # hkl # hkl The principal statistic used to evaluate the quality of structural models F O " F C F O Problem: unjustified fit parameters can be introduced to drive R arbitrarily low ( overfitting ) Solution: Remove a set (5-10%) of reflections from the data and exclude them from refinement ( cross validation ) R free = # hkl # hkl F O * " F C F O * Observations are sequestered in the test set and not included in the refinement The R free is highly correlated with model quality and can easily detect overfitting

8 How to Evaluate Crystallographic Models What is a good R factor? Should be below 30% in all cases Should be approx. 10x resolution limit for data D min <3.0 Å For atomic resolution data (D min <1.2Å), should always be <20% R free -R should be 5% or less What is a good Ramachandran plot? Should have less than 1% of residues in disallowed regions Should have more than 90% of residues in core regions What is good geometry? Bond length RMSD should be approx Å Angle RMSD should be approx. 1-2 No chirality deviations No close contacts (van der Waals violations) No planarity deviations (Phe, Trp, Tyr, His, A,T,G,C,U)

9 Traditional X-ray Sources: Rotating Anodes Produce X-rays by bombarding a metal anode with high energy electrons Produced X-rays have a fixed energy that depends on anode metal C: cathode W: window A: anode T: target R: rotor S: stator Rotation increases X-ray flux by dissipating heat

10 Modern X-ray Sources: Synchrotrons Confined electron beams moving in circular orbits at nearly the speed of light produce polychromatic X-rays ESRF; Grenoble, France Synchrotron radiation Synchrotron storage rings produce bright, tunable X-rays that allow data to be collected from difficult samples Intense X-rays require cryocooled samples to limit radiation damage

11 Future X-ray Sources: Free Electron Lasers FEL design: A straight synchrotron Single particle imaging with FEL light index_eng.html Produce femtosecond pulses of X-rays so intense that it converts sample to plasma Will be able to measure diffraction from single molecules, eliminating need for crystals

12 Photoelectron Generation and Radiation Damage Damage to crystal by X-ray beam Photoelectron trajectory and energy Beam center KeV 17.8 KeV Sanishvilli et al., PNAS, 108 (5), 6127 Radiation damage is sample, wavelength, dose, and temperature-dependent

13 Redox Proteins Present Challenges for X-ray Crystallography Issues Crystals illuminated with X- rays are highly reducing environments Redox-active groups are typically more sensitive to radiation damage (e.g. disulfides, cofactors, etc.) Metals absorb X-rays well, generate damage, and can themselves be reduced Current Solutions Some ongoing studies on including radical quenchers in buffers Minimize time, dose, temperature, wavelength(?) Avoid elemental absorption edges when choosing wavelength Note: Neutron diffraction suffers from none of these problems

14 Case Study: Isocyanide Hydratase

15 Isocyanides Are Electronically Interesting Note: carbon atom can be both nucleophile and electrophile Isocyanide hydratase (ICH) converts isocyanides to N-formamides in pseudomonads and possibly other organisms

16 ICH is a Member of the DJ-1 Superfamily 1. ICH is an obligate dimer with a highly conserved, catalytically essential cysteine residue (Cys101) 2. Structurally similar, but functionally unrelated, to DJ-1

17 Table 1: A Quiz Lakshminarasimhan et al., JBC 285, 38 (2010).

18 The Catalytic Cysteine is Oxidized in the ICH Crystal Structure WT ICH 1.05 Å resolution C101S ICH 1.00 Å resolution Likely a consequence of X-ray irradiation: beware of cysteine-sulfenic acids (Cys-SOH) in crystal structures

19 Mutating Cys101 Causes Structural Changes Black is WT ICH, Grey is C101A ICH Loss of a single hydrogen bond between Ile152 and Cys101 causes a major backbone shift

20 ICH Samples Multiple Conformations in the Crystal Black is WT IHC, Grey is C101A WT ICH Difference electron density (green) shows where atoms should be but are not present in the model 1. WT ICH natively samples a helix-shifted conformation in the crystal that is the dominant conformation for C101A ICH 2. Crystal structures are NOT static snapshots

21 Possible Mechanism for ICH Note that carbenoid form of the isocyanide is shown here, as it is an electrophile

22 Summary 1. Intense modern synchrotron X-ray sources provide ample opportunity for radiation damage 2. Redox active proteins are particularly vulnerable to radiation damage due to photoelectron reduction at metals, cysteines, and redox-active cofactors 3. Special precautions can limit, but not prevent, X-ray induced redox changes 4. The crystal structure of ICH shows evidence of photoreduction/oxidation at the active site cysteine 5. The electron density shows clear evidence of conformational polymorphism (i.e. not a static snapshot) 6. The crystal structure allows us to propose a testable mechanism for this unusual enzyme

23 Acknowledgements Current Members: Dr. Jiusheng Lin Peter Madzelan * Nicole Milkovic * Janani Prahlad Former members: Maha Lakshminarasimhan * Ruth Nan * Lauren Barbee * Synchrotron Data Collection APS, BioCARS 14BMC * Involved in the ICH project Funding: NIH (R01 GM092999), Redox Biology Center