A Primer in X-ray Crystallography for Redox Biologists

Mark Wilson

Karolinska Institute June 3rd, 2014

X-ray Crystallography Basics

http://en.wikipedia.org/wiki/X-ray_crystallography

Optimistic workflow for crystallography

Fourier Transform Fourier

Transform-1

Experiment Schematic

Monochromatic X-rays typically used

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/Rfree: A measure of model quality; fractional disagreement between model and

data

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 element-asymmetric unit

The collection of symmetry operations that generate the unit cell from the asymmetric unit compose the space group

http://www.rcsb.org/pdb/101/static101.do?p=education_discussion/Looking-at-Structures/ bioassembly_tutorial.html

Symmetry operation here is the two-fold

Why Crystallographers Worry About Phases

The amplitudes, but not phases, of the structure factors are experimentally measured

http://www.ysbl.york.ac.uk/~cowtan/fourier/magic.html

Fourier transform-1 Fourier transform

Duck |F|

Cat φ

Duck φ

Cat |F|

Problem: The phases, not the amplitudes, completely dominate the inverse transform

The data we need most are the data we cannot directly measure

Structures Are Models of Electron Density

Diffraction data (one of ~360 images) Electron density with model

Calculate density

Build model

Quantifying Model Quality: The R Value

!

R =

FO " FChkl#

FOhkl#

!

Rfree =

FO* " FC

hkl#

FO*

hkl#

Solution: Remove a set (5-10%) of reflections from the data and exclude them from refinement (“cross validation”)

Observations are sequestered in the “test” set and not included in the refinement

Problem: unjustified fit parameters can be introduced to drive R arbitrarily low (“overfitting”)

The principal statistic used to evaluate the quality of structural models

The Rfree is highly correlated with model quality and can easily detect overfitting

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 Dmin <3.0 Å •  For atomic resolution data (Dmin<1.2Å), should always be <20% •  Rfree-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. 0.01-0.02 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)

Traditional X-ray Sources: Rotating Anodes

http://en.wikipedia.org/wiki/X-ray_tube

C: cathode W: window A: anode T: target

R: rotor S: stator

•  Produce X-rays by bombarding a metal anode with high energy

electrons

•  Produced X-rays have a fixed energy that depends on anode

metal

•  Rotation increases X-ray flux by dissipating heat

Modern X-ray Sources: Synchrotrons

ESRF; Grenoble, France

Confined electron beams moving in circular orbits at nearly the speed of light produce polychromatic X-rays

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

Synchrotron radiation

Future X-ray Sources: Free Electron Lasers

http://mpsd.cfel.de/images/content/e101/e57509/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

FEL design: A “straight” synchrotron Single particle imaging with FEL light

Photoelectron Generation and Radiation Damage

Damage to crystal by X-ray beam

http://biop.ox.ac.uk/www/garman/projects.html

Radiation damage is sample, wavelength, dose, and temperature-dependent

Photoelectron trajectory and energy

14.4 KeV 17.8 KeV

Sanishvilli et al., PNAS, 108 (5), 6127

Beam center

Redox Proteins Present Challenges for X-ray Crystallography

•  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

•  Some ongoing studies on including radical quenchers in

buffers

•  Minimize time, dose, temperature, wavelength(?)

•  Avoid elemental absorption edges when choosing wavelength

Issues Current Solutions

Note: Neutron diffraction suffers from none of these problems

Case Study: Isocyanide Hydratase

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

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

Table 1: A Quiz

Lakshminarasimhan et al., JBC 285, 38 (2010).

The Catalytic Cysteine is Oxidized in the ICH Crystal Structure

Likely a consequence of X-ray irradiation: beware of cysteine-sulfenic acids (Cys-SOH) in crystal structures

WT ICH 1.05 Å resolution

C101S ICH 1.00 Å resolution

Mutating Cys101 Causes Structural Changes

Loss of a single hydrogen bond between Ile152 and Cys101 causes a major backbone shift

Black is WT ICH, Grey is C101A ICH

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”

Possible Mechanism for ICH

Note that carbenoid form of the isocyanide is shown here, as it is an electrophile

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

Acknowledgements Current Members:

Dr. Jiusheng Lin Peter Madzelan* Nicole Milkovic* Janani Prahlad

Former members: Maha Lakshminarasimhan*

Ruth Nan*

Lauren Barbee*

*Involved in the ICH project Funding: NIH (R01 GM092999), Redox Biology Center

Synchrotron Data Collection

APS, BioCARS 14BMC