Methods for Structure
Determination
Chemistry and Chemical Biology
Rutgers University
How are macromolecular
structures determined?
X-ray
(X-ray crystallography)
NMR
EM
(Nuclear Magnetic Resonance)
(Electron Microscopy)
Protein Data Bank
Download
X-ray Crystallography
Target selection
Protein production
Crystallization
Data collection
Phasing
Structure calculation
Refinement
Validation
Deposition to PDB
Annotation
http://www-structmed.cimr.cam.ac.uk/Course/Overview/Overview.html#methods
Nuclear Magnetic Resonance
(NMR)
http://www.embl.de/nmr/sattler/teaching
Electron Microscopy (EM)
Courtesy, Cathy Lawson, Rutgers University
The Data Pipeline
Genomic
Based Target
Selection
Isolation,
Expression,
Purification,
Crystallization
Data
Collection
Structure
Determination
PDB Deposition
& Release
3D Models
Annotations
Publications
X-ray Crystallography
•
•
•
•
Crystals
Symmetry
Diffraction
X-ray Crystallography
Mineral
Crystals
Two-Dimensional “Crystal”
Translational Symmetry
M.C. Escher
Crystal Lattice
Crystal Lattice
Alexander McPherson, Introduction to Macromolecular Crystallography Wiley-Liss, 2002
The Unit Cell
Symmetry
Two-Dimensional “Crystal” With
Mirror Symmetry
M.C. Escher
Crystal Symmetry
Crystal Systems
Jenny Pickworth Glusker, Kenneth N.
Trueblood, Crystal Structure Analysis: A
Primer, Oxford University Press, 1985
The International Tables
The Fourier
Duck
Light Diffraction
Henry S. Lipson Crystals and X-rays Taylor & Francis 1970
Fourier Analysis in Microscopy
Why Use X-rays?
http://bccp.lbl.gov/Academy/wksp_pix_1/spectrum.gif
X-ray Diffraction
Gale Rhodes, Crystallography Made Crystal Clear: A Guide for Users of Macromolecular Models,
Academic Press, 1993
X-ray Crystallography
X-ray Diffraction Photograph
Rosalind Franklinʼs diffraction photo of DNA
Another X-ray Photo
Myoglobin
Hemoglobin
Lysozyme
Ribonuclease
Crystal
Structures
Myoglobin: Kendrew, Bodo, Dintzis, Parrish,
Wyckoff, Phillips, Nature 181 662-666, 1958.
Hemoglobin: Perutz, Proc. R. Soc. A265,
161-187,1962. Lysozyme: Blake, Koenig,
Mair, North, Phillips, Sarma, Nature 206 757,
1965. Ribonuclease: Kartha, Bello, Harker,
Nature 213, 862-865 1967. Wyckoff,
Hardman, Allewell, Inagami, Johnson,
Richards. J. Biol. Chem. 242, 3753-3757,
1967.
The Steps in the Process
•  Isolation,
•  Expression,
Target •  Purification,
Data
Selection
•  CrystallizationCollection
Structure Structure Functional Solution Refinement Annotation
Publication
Protein Preparation
Cloning
Expression
Purification
Crystallization Method
Common precipitants:
– Polyethylene glycol
– Salts
•  ammonium sulfate
•  sodium chloride
–  Alcohols
•  Isopropanol
•  Methylpentanediol (MPD)
Cover
Slip
Precipitant
Solution
Protein
+
Precipitant
Crystallization Techniques
Hampton Screen
Results
isopropanol
PEG
Ammonium sulfate
Sodium phosphate
4 crystal forms of E. coli trp repressor
Crystal Mounting for Diffraction Trials
•  Transfer crystal briefly to cryoprotectant solution
–  Xtalln solution + glycerol, PEG, sugars, salts, or
alcohols
•  Scoop up crystal with nylon loop
•  Plunge into liquid nitrogen (LN2)
•  Store in LN2
•  Labeling/tracking very important!
Crystal mounting movie: http://www.youtube.com/watch?v=J4OD_b9XKh4
Data Collection Instrumentation
–  X-ray generation (decelerating electrons)
–  Optics to select single wavelength
–  Crystal position control (centering, precise rotation)
–  Detector
–  Beamstop
–  Gas N2 stream (100oK)
Rotating Anode Diffractometer
Synchrotron X-ray source
http://www.nsls.bnl.gov
Synchrotron Beamline Diffractometer
NSLS
Beamline
X12C
Bragg’s Law
nλ = 2d sin θ
2θ
angle
between
incident
and
reflected
beams
d
spacing
between
planes
λ
Wavelength
n
order
of
diffraction
http://www.bmsc.washington.edu/people/merritt/bc530/bragg/
try the Java Applet!
Crystal Diffraction
High Resolution
(large angle)
Water Ring
~3-5 Å
Beam Stop
Shadow
Low Resolution
(small angle)
Jeff Dahl, Sars protease, http://en.wikipedia.org/wiki/File:X-ray_diffraction_pattern_3clpro.jpg
Different crystal forms of the same protein
yield different diffraction patterns
trp repressor, sodium phosphate
trp repressor, ammonium sulfate
Action!
Crytal Diffraction: http://ucxray.berkeley.edu/~jamesh/movies/
Data Collection Strategy
•  The American Method
– shoot first, ask questions later
•  Special cases:
– Radiation Decay
– High mosaicity
– Anisotropy
Data Obtained
•  Crystal unit cell
dimensions
•  Lattice type, possible
space groups
•  Resolution Limit
•  Merged data set with
index, intensity + error
for each reflection
a
=
36.67
Å
b
=
79.39
Å
c
=
39.97
Å
α
=
90.0°
ß
=
91.25°
γ
=
90.0°
Monoclinic lattice (P2 or P21)
H
K
0
0
0
0
0
0
0
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
...etc.
L
12
18
24
30
3
4
5
6
7
8
9
10
11
12
13
14
intensity error
6714.3
347.2
-8.9
16.3
979.5
62.4
4136.4
272.5
3035.4
70.2
0.0
0.7
0.1
0.6
838.4
20.4
14903.0
535.6
2759.4
64.7
1403.5
31.0
109.4
5.6
31739.5 1611.5
231.9
7.6
5433.0
94.3
12392.7
211.4
Electron Density Calculation
•  The electron density at (x,y,z) is the sum of
contributions of all structure factors Fhkl
Ρ(x,y,z) = Σ F hkl e -2πi (hx + ky +lz)
Structure Factor
(complex number)
•  The electron density provides an image that is
averaged :
•  over all molecules in the crystal
•  over the time of the diffraction experiment
Phase Problem
•  The complete Structure Factor F for a
reflection includes the phase, which cannot
be measured directly.
F hkl = |F hkl| e -iϕhkl
Structure Factor
Amplitude:
from experimental
measurements
Phase:
must be estimated
Phasing Methods
•  Direct Phasing
–  Isomorphous replacement
–  Anomalous Dispersion
•  Molecular Replacement
Direct Phasing
•  Add or incorporate atoms yielding small
changes in reflection amplitudes
•  Can estimate phase by solving for heavy
(or anomalous atom) substructure
Anomalous Phasing
•  MAD:
–  Multiple wavelength anomalous dispersion
–  F+ vs. F- and X-ray wavelength dependence
•  SAD
–  Single wavelength anomalous dispersion
–  F+ vs. F- at single wavelength
•  Selenium, Bromine, Mercury…
Molecular Replacement (MR)
•  New structure expected to resemble one
previously determined
•  Use Patterson-based methods to find
the orientation of known model in new
crystal lattice
Orient
Adapted from Acta Cryst D Aug. 2006 cover image
Electron Density Map
Fit model to map
Structure Assessment:
the R Factor
Model Building-Refinement
Cycle
Initial Model
Refine
Experimental
Data
Stereochemical
Knowledge
Calculate
Map
Edit model
Final
Model
Structural Data
-snip-
PDB 3a6b
Typical
Statistical
Table
Validation: Ramachandran Plot
Model Improvement
OLD MODEL
Resolution limit = 3.1 Angstrom
NEW MODEL
Resolution limit= 2.1 Angstrom
The Movie Links
•  Crystal Mounting Robot
–  http://www.youtube.com/watch?v=J4OD_b9XKh4
•  Crystal Diffraction
–  http://ucxray.berkeley.edu/~jamesh/movies/
Enjoy!