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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
h k l I σ(I) 0 0 2 3523.1 91.30 0 3 -1.4 2.80 0 4 306.5 9.60 0 5 -0.1 4.70 0 6 10378.4 179.8 . . .
? Phase Problem ?
MIRMADMR
Electron density:(x y z) = 1/V |F(h k l)| exp[–2i (hx + hy + lz) + i(h k l)]
Bragg’s
law
Fourier transform
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-raysWhy don’t we just use a microscope to look at proteins?
• Size of objects imaged limited by wavelength. Resolution ~ /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
X-ray beam
crystal
Film/Image plate/CCD camera
~1015 molecules/crystalDiffraction pattern is amplified
Bragg’s Law:2d sin = n
End result – really!Fourier transform of diffraction spots electron density fit a.a. sequence
Protein
DNA pieces
(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 resolutionMore 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, often2-3 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:
X-rayNMR
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
• 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
Function from structure
Domain organization
• Large proteins have polypeptide regions that fold in isolation
• May have distinct functional roles – Example:
glyceraldehyde-3-phosphate 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
Class Folds Superfamilies Families
All 171 286 457
All 119 234 418
Alpha & beta () 117 192 501
Alpha & beta () 224 330 532
Multi-domain proteins 39 39 50Membrane /cell-surface proteins 34 64 128
Small proteins 61 87 135
Total 765 1232 2164
Structural Classification of Proteins18946 PDB Entries, 49497 Domains (1 March 2002)
(excluding nucleic acids and theoretical models)
http://scop.berkeley.edu/ or http://scop.mrc-lmb.cam.ac.uk/scop/
Have all folds been found?
Red = Old foldsBlue = New folds