Macromolecular Small and Wide-Angle Fiber Diffraction with Synchrotron Radiation

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Macromolecular Small and Wide-Angle Fiber
Diffraction with Synchrotron Radiation

• Tom Irving
• BioCAT, Dept. BCPS and CSRRI
• Illinois Institute of Technology

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Scope of Lecture

• Why do Fiber Diffraction?
• Physical Principles
• Experimental methods
• Applications
• Advantages of Third Generation Synchrotrons for
  Fiber Diffraction

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References

• Basics C. Cantor and P. Schimmel Biophysical
  Chemistry part II Techniques for the study of
  Biological Structure and Function Chapter 14.
  Freeman, 1980
• A terrific introduction to helical diffraction
  theory and muscle diffraction John Squire The
  Structural Basis of Muscular Contraction Plenum,
  1981
• The classic workB.K. Vainshtein Diffraction of
  X-rays by Chain Molecules Elsevier, 1966.

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More references

• Good introduction to Fiber crystallography
• Chandrasekaran, R. and Stubbs, G. (2001). Fiber
  diffraction. in International Tables for
  Crystallography, Vol. F Crystallography of
  Biological Macromolecules (Rossman, M.G. and
  Arnold, E., eds.), Kluwer Academic Publishers,
  The Netherlands, 444-450.

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Why Fiber Diffraction ?

• Atomic level structures from crystallography or
  NMR gold standard for structural inferences
• Large class of fibrous proteins
• Actin, myosin, intermediate filaments,
  microtubules, bacterial flagella, filamentous
  viruses, collagenous connective tissue
• Difficult to crystallize but can be induced to
  form oriented assemblies
• Some systems such as muscle and tendon naturally
  form such assemblies

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Fibers Usually Hexagonally Packed
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Hexagonal Lattice
u
y
x
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Ewald Sphere
k a
2 ?
1
h a
?
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Fiber Pattern Insect Muscle
Equator
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Fiber Cross-section
Diffraction
X-rays
Complete Statistical rotation 1,0 1,0 0,1
0,1 1,1 1,1 1,1 1,1
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A
B
C
Ordering in Fibers A - Crystalline fiber B
Semicrystalline Fiber C Non-crystalline fiber
ltI(s)gt ltFm(S)FL(S)2gt
Average over all molecular and lattice
orientations
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Fiber Pattern Insect Muscle
Equator
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Geometry of Fiber Patterns
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Fibrous Proteins Usually Show Helical Symmetry
P pitch p subunit axial translation
distance R true repeat distance
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Diffraction from a Continuous Helix
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Bessel Functions and Diffraction from a Helix
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Rosalind Franklins Pattern from B-DNA
Franklin Gosling, 1953Nature 171740
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Diffraction from a Discontinous Helix
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Discontinuous Helix with Non-integral
Subunits/Turn
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Helical Selection Rule

• Bessel functions that contribute to a given layer
  line depend on helical parameters
• For a helix of pitch P with an axial subunit
  displacement of p
• and u subunits in t turns of a helix
• Allowed Bessel functions (of order n) on layer
  line l are
• l mu nt
• m is an integer indicating successive meridional
  reflections

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Crystals of Helical Molecules
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Crystalline Forms of DNA
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Multi-Stranded Helices
If N strands Only every Nth Layer -line allowed
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Fiber Diffraction Often Just Used to Find Gross
Molecular Parameters

• In many cases one can make structural inferences
  without a full-blown structure solution
• Helical parameters in Polyamino-acids and nucleic
  acids
• Topology of viruses and other large molecular
  complexes
• Test hypotheses concerning influence of
  inter-filament lattice spacing

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Rosalind Franklins Pattern from B-DNA
M
Layer lines (L) separated by 34 Å nm Meridional
(M) reflection at 3.4 Å gt 10 residues/turn
L
M
Franklin Gosling, 1953 Nature 171740
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Diffraction from Poly L-Alanine -?-helix
1.5 Å/residue Pitch 5.4 Å 18 residues/5
turns Layer lines every (5.4 l/5)-1 l18m5n
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Sometimes a modeling approach is taken
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Fiber Crystallography

• Most fiber structures result of model building
  studies
• There have been a relatively small number of
  Fiber structure solutions.
• TMV by Caspar and others
• High resolution structure by Keichi Namba on
  bacterial flagella (Yamashita et al., 1998 Nature
  SB) aligned by high magnetic fields
• Orgel et al. (2001 Structure) -first MIR
  structure of a natural fiber - Type I Collagen
  from rat tail

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Tobacco mosaic virus
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Fiber diffraction pattern of gold-labeled rat
tail tendon Collected native, gold, iodide
patterns Orgel et al. Structure 91-20, 2001
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Background subtracted image of row lines showing
closely -spaced but resolved diffraction spots
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Two unit cells of the fiber structure of rat tail
(Type I) collagen. 67 nm long axis has been
compressed 12 x for display purposes Map is 20 Å
wide Resolution is 5.4 Å vertically and 10 Å
horizontally
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Muscle Diffraction
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Hierarchy of Muscle Structure
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Muscle Filament Substructure
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Sliding Filament Theory of Muscle Contraction
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Muscle Filament Lattice
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Layer Line Pattern From Striated Muscle
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Relaxed
Isometric
After quick release
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Cardiac Muscle
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Synchrotrons and Fiber Diffraction

• Early work all done with conventional sources -
  why need synchrotron?
• Patterns weak, have high backgrounds, frequently
  have multiple closely spaced lattices
• Studies benefit from greatly increased beam
  quality
• Greatly increased flux permits time-resolved
  experiments

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Undulator Beamline Performance

• Total X-ray flux 1-2.5 x1013 photons/s
• Focal spot size ranges from lt 40 µm vertical and
  lt 200 µm horizontal to 3 x 1.5 mm
• First order resolution gt 1500 Å
• Order to order resolution gt 10000 Å

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diffraction pattern from oriented
sol odontoglossum ringspot virus BioCAT 2001
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Functional integration time resolved
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Diffraction features very similar in all insect
muscles Layer lines orders of 232 nm long
repeat Split 14.5 and 7.2 nm reflections from
axial stagger of filaments in flies. Note First
row line spot on 19.3 nm layer line

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Fly Flight simulator

• Visual field simulator
• Wing-beat phase trigger
• Rapid shutter

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Analysis Software

• Rate limiting step is data analysis
• Long tradition of rolling your own
• CCP 13 project http//www.ccp13.ac.uk
• Comprehensive data extraction suite
• Complementary NSF RCN Stubbs (Vanderbilt) PI
  will add angular deconvolution, other features to
  suite