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DNA/Protein structure-function analysis and prediction
• Protein Structure Determination:
– X-ray Diffraction(Titia Sixma, NKI)
– Electron Microscopy/Diffraction(Titia Sixma, NKI)
– NMR Spectroscopy(Lorna Smith, Oxford)
– Other Spectroscopic methods
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Spectroscopy: The whole spectrum
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DNA/Protein structure-function analysis and prediction
• Protein Structure Determination:
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
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Structure determination method X-ray crystallography
Purified protein
Crystal
X-ray Diffraction
Electron density
3D structureBiological interpretation
Crystallization
Phase problem
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Protein crystals• Regular arrays of protein molecules
• ‘Wet’: 20-80% solvent• Few crystal contacts
• Protein crystals contain active protein• Enzyme turnover• Ligand binding
Example of crystal packing
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Examples of crystal packing
2 Glycoprotein I~90% solvent (extremely high!)
Acetylcholinesterase~68% solvent
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Problematic proteins• Multiple domains
• Similarly, floppy ends may hamper crystallization: change construct
• Membrane proteins
• Glycoproteins
Flexible
Lipid bilayer
hydrophilic
hydrophilic
hydrophobic
Flexible and heterogeneous!!
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Experimental set-up• Options for wavelength:
– monochromatic, polychromatic – variable wavelength
Liq.N2 gas stream
X-ray source
detector
goniometer
beam stop
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Diffraction imageDiffraction image
Water ring
Diffuse scattering (from the fibre loop)
reciprocal lattice reciprocal lattice (this case hexagonal)(this case hexagonal)
Beam stop
Increasing resolution
Direct beam
ReflectionsReflections ( (h,k,lh,k,l) ) withwith I( I(h,k,lh,k,l))
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The rules for diffraction: Bragg’s law
• Scattered X-rays reinforce each other only when Bragg’s law holds:
Bragg’s law: 2dhkl sin = n
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Phase Problem• If phases hkl and structure factor F(hkl) known:
• compute the electron density (x,y,z)
– In the electron density build the atomic 3D model
• However, the phases hkl are unknown !
hklhkl
hkl
lzkyhxiihklFV
lzkyhxihklFV
zyx
)}(2exp{exp)(1
)}(2exp{)(1
),,(
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FH, HFK, K
FH, KFK, H
How important are these phases ??• Fourier transform photo’s
of Karle (top left) and Hauptman (top right) (two crystallography pioneers)
• Combine amplitudes FK with phase H and inverse-fourier transform
• Combine amplitudes FH with phase K and inverse-fourier transform
(Taken from: Randy J. Read)
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How can we solve the Phase Problem ?• Direct Methods
– small molecules and small proteins– needs atomic resolution data (d < 1.2 Å) !
• Difference method using heavy atoms– multiple isomorphous replacement (MIR)– anomalous scattering (AS)– combinations (SIRAS,MIRAS)
• Difference method using variable wavelength– multiple-wavelength anomalous diffraction (MAD)
• Using a homologous structure– molecular replacement
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Building a protein model• Find structural elements:
-helices, -strands• Fit amino-acid sequence
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Building a protein model• Find structural elements:
-helices, -strands• Fit amino-acid sequence
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Effects of resolution on electron density
Note: map calculated with perfect phases
d = 4 Å
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d = 3 Å
Effects of resolution on electron density
Note: map calculated with perfect phases
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d = 2 Å
Effects of resolution on electron density
Note: map calculated with perfect phases
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d = 1 Å
Effects of resolution on electron density
Note: map calculated with perfect phases
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Refinement process
• Bad phases poor electron density map
errors in the protein model
• Interpretation of the electron density map improved model
improved phases improved map
even better model
… iterative process of refinement
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Validation• Free R-factor (cross validation)
– Number of parameters/observations
• Ramachandran plot • Chemically likely (WhatCheck)
– Hydrophobic inside, hydrophilic outside
– Binding sites of ligands, metals, ions
– Hydrogen-bonds satisfied– Chemistry in order
• Final B-factor values
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DNA/Protein structure-function analysis and prediction
• Protein Structure Determination:
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
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Electron microscopy• Single particle
– Low resolution, not really atomic– Less purity of protein, more transient state analysis
• Two-dimensional crystals– Suited to membrane proteins
• Fibres– Acetylcholine receptor– Muscles, kinesins and tubulin
• Preserve protein by– Negative stain (envelope only)– Freezing in vitreous ice (Cryo-EM, true density maps)
• High resolution possible but difficult to achieve– For large complexes: Combine with X-ray models
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Electron diffraction from 2D crystals: Nicotinic Acetylcholine Receptor
Unwin et al, 2005
G-protein coupled receptors
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Electron diffraction: near atomic resolution• Structure of the alpha beta tubulin dimer by electron
crystallography. Nogales E, Wolf SG, Downing KH.Nature 1998 391 199-203
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Select particles Sort into classes
Average Reconstruct 3D image
Single particle Electron Microscopy
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17 Jan 2006 Leiman et al. Cell. 2004 Aug 20;118(4):419-29
Cryo EM reconstruction:Tail of bacteriophage T4
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DNA/Protein structure-function analysis and prediction
• Protein Structure Determination:
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
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1D NMR spectrum of hen lysozyme (129 residues)
• Too much overlap in 1D: 2D
• 1H-1H• 1H-15N• 1H-13C
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N
C
C
N
C
C
H
O
N
H
O
H C H
R H
HHC
Amino acid spin system
C
O
Amino acid spin system
Step 1: Identification of amino acid spin systems
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Val
Val
Thr
Thr
2D COSY spectrum of peptide in D2O
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N
C
C
N
C
C
H
O
N
H
O
H
C H
R H
HHC
NH(i)-NH(i+1) NOE
H(i)-NH(i+1) NOE
H(i)-NH(i+1) NOE
NH(i)-NH(i+1) NOE
Step 2: Sequential assignment
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Gly
Gly
AspAsn
Asp
Phe
ThrSer
Leu
Val
2D NOESY spectrum
• Peptide sequence (N-terminal NH not observed)• Arg-Gly-Asp-Val-Asn-Ser-Leu-Phe-Asp-Thr-Gly
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Nitroreductase Dimer: 217 residues• Too much
overlap in 2D: 3D
• 1H-1H-15N• 1H-13C-15N
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Structural information from NMR: NOEs• For macromolecules such as proteins:
– Initial build up of NOE intensity 1/r6
– Between protons that are < 5Å apart
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O
N
H
O
N
H
R
H
O
N
O
N
H
O
R
HN
H
O
N
H
R
H
H
H
R
O
N
H
H
R
R
H
R
H
O
N
H
OH
R
H
R
NOEs in -helices• NH-NH(i,i+1) 2.8Å• H-NH(i,i+3)3.4Å• H-H(i,i+3) 2.5-4.4Å
• Cytochrome c552 3D 1H-15N NOESY
– H-NH(i,i+3)– resides 38-47– form -helix
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NN
NN
R
O R
O R
OH
H
H
H
R
OH H
H H
NN
NN
N
R
OR
OR
O H
H
H
H
R
OHH
HH
O
H
NOEs in -strands• H-NH(i,j) 2.3Å• H-NH(i,j) 3.2Å
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Long(er) range NOEs • Provide information about
– Packing of amino acid side chains – Fold of the protein
• NOEs observed for CH of Trp 28 in hen lysozyme
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N
C
C
N
C
C
H
O
N
H
O
H C H
R H
HHC
C
O
Spin-spin coupling constants• Fine structure in COSY cross peaks• For proteins 3J(HN.H) useful:
– Probes main chain torsion angle
• Hen lysozyme:
2
3
4
5
6
7
8
9
10
0 20 40 60 80 100 120 140
Co
up
ling
co
nst
an
t (H
z)
Residue number
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Structural information from NMR3. Hydrogen exchange rates
• Dissolve protein in D2O and record series of spectra
– Backbone NH (1H) exchange with water (2H)• Slow exchange for NH groups
– In hydrogen bonds– Buried in core of protein
After 20 mins After 68 hours 1H-15N HSQC spectra for SPH15 in D2O
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Structural information from NMR• NOEs
– 1H - 2H 1.8-2.5Å (strong)– 2H - 40HN 1.8-5.0Å (weak)– 3H - 88H2 1.8-5.0Å (weak)– 3H - 55H 1.8-3.5Å (medium)
• Spin-spin coupling constants– 1C-2N-2C-2C -120+/-40°– 4C-5N-5C-5C -60+/- 30°– 5C-6N-6C-6C -60+/- 30°
• Hydrogen exchange rates– 10HN-6CO 1.3-2.3Å 10N-6CO 2.3-3.3Å– 11HN-7CO 1.3-2.3Å 11N-7CO 2.3-3.3Å
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NMR structure determination: hen lysozyme
• 129 residues– ~1000 heavy atoms– ~800 protons
• NMR data set– 1632 distance restraints– 110 torsion restraints– 60 H-bond restraints
• 80 structures calculated• 30 low energy
structures used
0
2000
4000
6000
8000
1 10 4
1.2 10 4
10 20 30 40 50 60 70
Tot
al e
nerg
y
Structure number
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Solution Structure Ensemble• Disorder in NMR ensemble
– lack of data ?
– or protein dynamics ?
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DNA/Protein structure-function analysis and prediction
• Protein Structure Determination:
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
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Ultrafast Protein Spectroscopy• Structure-sensitive technique• State of protein and substrate
– Redox state– Protonation– Elektronen
• Follow reactions in real-time• Why ultrafast spectroscopy?
– Molecular movement: time scales of 10 fs – 1 ps (10-15 – 10-12 s)
– O H stretching frequency: ~3500 cm-1 (~ 10-14 s)• Transfer or movement of proton, electron or C-atom
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1750 1700 1650 1600
-2
0
2
mO
D
Wavenumber (cm-1)
Chl*/Chl in THF Pheo*/Pheo in THFketo
ester
Keto and ester C=O in Chlorophyll a, Pheophytin a are redox and environmental probes
Excited state difference spectra of Chl and Pheo
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X – C – O – H
O – XOω1
ω2
ω1’
ω2’
Why is vibrational spectroscopy sensitive to structure?
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H-bond response during the photocycle
1780 1760 1740 1720 1700 1680-1.0
-0.5
0.0
0.5
mO
D
Wavenumber (cm-1)
2 ps, excited state 700 ps, I0 inf, I1
1740 1720 1700 1680 1660 1640
-1
0
1
2
mO
D
Wavenumber (cm-1)
1.5 ps, excited state 800 ps, I0 inf, I1
S
NHOO
O HO
H
O
O
H
NH
H2N NH2
Cys69
Thr50
Tyr42
Glu46
+
Arg52
Replace Glu with Gln whichdonates weaker H-bond
weaker stronger
broken
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49
Str
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17 Jan 2006
Summary
Method Pro Con
X-ray crystallography
High resolution
No size limits
Easy addition of ligands
Crystals required
Phase problem
Electron microscopy /
diffraction
Single particles possible
Well suited to membrane proteins
Labour intensive
High resolution difficult
NMR spectroscopy
In solution
Dynamic information possible
Resolution variable
Limited size (< ~30kDa)
Other spectroscopy
(Very) high time resolution (ps!)
Very sensitive
Technically extremely complex
Limited applicability
Limited information