T H E U N I V E R S I T Y of T E X A S
SCHOOL OF HEALTH INFORMATION
SCIENCES AT HOUSTON
X-Ray Crystallography Pt. II
For students of HI 6001-125
Computational Structural Biology
Sugoto Chakravarty, Ph.D.
Baylor College of Medicine
http://biomachina.org/courses/structures/03.html
Periodic Array of 2N+1 Identical Atoms
Atomic Form Factors
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Periodic Array of 2N+1 Identical Atoms
Fringe Function and Diffraction
Condition for Diffraction Maximum
Condition for Diffraction Maximum
Miller Indices in a Lattice
Two Conditions for Diffraction
Two Conditions for Diffraction
Visualizing the Two Conditions for Diffraction
Diffraction Pattern
Observable Part of Ewald Sphere
Scattering in 3 Dimensions
Argand Diagram of Structure Factors
Crystal Lattice and Convolution
(x-a)
Crystal Lattice and Convolution
Unit Cell and Symmetry
Unit Cell Types Determined by Symmetry
7 Crystal Systems and 14 Bravais Lattices
Different Lattices and Centering
Triclinic &
Monoclinic
Orthorhombic
Tetragonal
Cubic http://www.uwgb.edu/dutchs/
symmetry/bravais.htm
Applying a Screw Operation
Example of a Space Group
Braggs Law of Diffraction
Phase Solution
(Note: A.s. not explained here, similar to I.r., but using special wavelengths to break Friedel
symmetry, see http://www.bmsc.washington.edu/scatter/AS_tutorial.html)
Isomorphous Replacement
Patterson Functions to Locate Heavy Atoms
Patterson are Inter-Atomic Distance Maps
Pattersons Determine Heavy Atom Vectors
Patterson Heavy Atom Peaks May
Solve Structure
Single Isomorphous Replacement (SIR)
Resolving Phase Ambiguities in SIR
Multiple Isomorphous Replacement (MIR)
Errors in MIR Phases
Errors in MIR Phases
Errors in MIR Phases
Errors in MIR Phases
Errors in MIR Phases
Best MIR Phase
contd
Best MIR Phase
Figure of Merit of a Phase
Molecular Replacement
Similar structure exists (sequence identity)
MIR not required
Orient the known structure as closely as
possible to the unknown structure
Place the known structure as correctly as
possible
Rotational and translational parameters will
give a good set of starting phases
Molecular Replacement
Rotation Conventions
Euler
Rossman
Patterson Rotation Functions
Orientation of Unknown Molecule
Position of Molecule: Translation Function
Intra- and Inter-Molecular Vectors
Another Translation Function
Averaging Using Inherent Symmetry
Unit cell
Particle + unit cell
symmetry
Symmetry preserved in
diffraction space
No of faces : 12 (5-folds)
No of edges : 30 (2-folds)
No of vertices : 20 (3-folds)
T=3 Icosahedron
Averaging
Noise
25 2 35
30 5 34
26 1 29
Amplitude
26 7 32
Signal
20 0 31
28 2 32
Position 19 3 25
20 4 28
Averaging and Iterative
Improvement of Phases
FT Noisy initial Average
Amplitudes + Cleaner atomic
initial phases Atomic map map
FTINV
Better phases FT
Average
(Combine with Better atomic
Amplitudes) maps
Poor Initial Phases
Improved Phases after Cycles of Averaging
Display of Electron Density
Refinement of Atomic Model
Refinement is Like Curve Fitting
Normal Equations in Refinement
Conjugate Gradient
Full LSQ Refinement not Possible
Anisotropic B-valuesThermal Ellipsoids
Bisotropic = const * <u2>
http://www.crystalimpact.com/diamond/v2feature-ellipsoids.htm
Rfree in Refinement of Atomic Models
Q: How good does this model predict the data
that it has not seen?
Curve fitting analogy: Does a 4th pt lie on a 3-pt
quadratic curve?
Rfree: Choose a random 5-10% test data sub-set
and calculate R-factor for this test set.
If the model is good, Rfree should closely follow
R of the remaining data set.
Refined Electron Density
Trp
Poorly defined ed
Lack of clear density
Uninterpreted Conformers in
density Lys, Asn
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Manual Editing Using Graphics
Disordered Lysine
Manual model editing is necessary
Concluding Remarks
- X-ray diffraction may be analyzed by
FT/series. Alternative methods also reported
- Phases may be determined using several
methods
- Phases may be improved by averaging and/or
refining the atomic model with model fitting
- Well-refined models are essential to correctly
interpret biological functions
Resources
Cantor, R.C. & Schimmel, P.R. (1980). Biophysical Chemistry Part II:
Techniques for the study of biological structure and function. New York; W.H.
Freeman & Co, Chapter 13.
Blundell, T.L. & Johnson, L.N. (1976). Protein Crystallography. London;
Academic Press.
Stout, G.H. & Jensen, L.M. (1968). X-ray Structure Determination: A
Practical Guide. New York: Macmillan
