Download 1. Overview

X-ray crystallography – an overview
(based on Bernie Brown’s talk, Dept. of Chemistry, WFU)
• Protein is crystallized (sometimes low-gravity
atmosphere is helpful e.g. NASA)
• X-Rays are scattered by electrons in molecule
• Diffraction produces a pattern of spots on a film that
must be mathematically deconstructed
• Result is electron density (contour map) – need to
know protein sequence and match it to density
• Hydrogen atoms not typically visible (except at very
high resolution)
X-ray Crystallography – in a nutshell
REFLECTIONS
Bragg’s
law
h k l
0
0
0
0
0
.
.
.
0
0
0
0
0
I
σ(I)
2 3523.1
3 -1.4
4 306.5
5 -0.1
6 10378.4
91.3
2.8
9.6
4.7
179.8
Fourier
transform
? Phase Problem ?
MIR
MAD
MR
Electron density: r(x y z) = 1/V
SSS |F(h k l)| exp[–2pi (hx + hy + lz) + ia(h k l)]
Crystal formation
• Start with supersaturated solution
of protein
• Slowly eliminate water from the
protein
• Add molecules that compete with
the protein for water (3 types:
salts, organic solvents, PEGs)
• Trial and error
• Most crystals ~50% solvent
• Crystals may be very fragile
Visible light vs. X-rays
Why don’t we just use a microscope to look at proteins?
• Size of objects imaged limited by
wavelength. Resolution ~ l/2
– Visible light – 4000-7000 Å (400-700 nm)
– X-rays – 0.7-1.5 Å (0.07-0.15 nm)
• It is very difficult to focus X-rays (Fresnel lenses)
• Getting around the problem
– Defined beam
– Regular structure of object (crystal)
• Result – diffraction pattern (not a focused image).
Diffraction pattern – lots of spots
Bragg’s Law:
2d sinq = nl
X-ray beam
crystal
~1015 molecules/crystal
Diffraction pattern is
amplified
Film/Image plate/CCD camera
End result – really!
Fourier transform of diffraction spots  electron density  fit a.a. sequence
DNA
pieces
Protein
(Dimer of
dimers)
Interference of waves
• In crystallography,
get intensity
information only, not
phase information
• Need to deconvolute
and obtain phase
information:
• THE PHASE
PROBLEM
How to get from spots to structure?
• Fourier synthesis
• Getting around phase problem
– Trial and error
– Previous structures
– Heavy atom replacement – make a landmark
– Ex: Selenomethionine
• Plenty of computer algorithms now
Electron density with incorrect phases
• Red is true
structure
The effect of resolution
More extensive diffraction pattern gives more structural
information = higher resolution
• 6.0-4.5 Å – secondary
structure elements
• 3.0 Å – trace polypeptide
chain
• 2.0 Å – side chain, bound
water identification
• 1.8 Å – alternate side chain
orientations
• 1.2 Å – hydrogen atoms
With computational tools,
spots become density
Flexible regions give smeared density, often
2-3 conformations visible, more than that invisible
Density becomes structure
Need to know protein sequence to trace backbone
Co-crystal structures
• Because of relatively high solvent content,
can often “soak in” substrate
• Then can solve structure of protein with
substrate bound
• If crystal cracks, good sign that substrate
binding or enzyme catalysis results in
conformational change in protein
• No longer has same crystal arrangement
NMR vs. crystallography
• Useful for different samples
• Generally good agreement
• E. coli thioredoxin:
NMR
X-ray
Note missing region
Known protein structures
• ~17,000 protein structures since 1958
• Common depository of x,y,z coordinates:
Protein data bank (http://www.rcsb.org)
• Coordinates can be extracted and viewed
• Comparisons of structures allows identification
of structural motifs
• Proteins with similar functions and sequences =
homologs
Growth in structure
determination
Function from structure
• Might identify a pocket lined
with negatively-charged
residues
• Or positively charged
surface – possibly for
binding a negatively
charged nucleic acid
• Rossmann fold – binds
nucleotides
• Zinc finger – may bind DNA
Domain organization
• Large proteins have
polypeptide regions that
fold in isolation
• May have distinct
functional roles
– Example:
glyceraldehyde-3phosphate
dehydrogenase
Protein families
• Similar function and overall structure
• But amino acid sequence may or may not be
highly conserved
• Limited number of protein domains
• Homologs versus structural motifs
SCOP Classification Statistics
Structural Classification of Proteins
18946 PDB Entries, 49497 Domains (1 March 2002)
(excluding nucleic acids and theoretical models)
Class
Folds
All a
All b
Alpha & beta (a/b)
Alpha & beta (a+b)
Multi-domain proteins
Superfamilies
Families
Membrane /cell-surface proteins
171
119
117
224
39
34
286
234
192
330
39
64
457
418
501
532
50
128
Small proteins
Total
61
765
87
1232
135
2164
http://scop.berkeley.edu/
or
http://scop.mrc-lmb.cam.ac.uk/scop/
Have all folds been found?
Red = Old folds
Blue = New folds