Understanding biology through structures Course work 2006 Structure Determination and Analysis :...

Understanding biology through structures Course work 2006

Structure Determination and Analysis : X-ray Crystallography


X-rays Intensity of diffracted beam

Diffraction

Source Sample Detector


Energy = hc λ


X-rays

•It is possible to translate information in the diffraction pattern into atomic structure using Bragg’s law, which predicts the angle of reflection of any diffracted beam from specific atomic planes

•Unlike using a light microscope, there is no way of re-focusing diffracted x-rays.

•Instead we must collect a diffraction pattern (spots).


A typical crystallography experimentPure protein

Grow crystal

Characterize crystals

Collect diffraction data

Solve phase problem

Calculate electron density map

Build/rebuild model

Refine model

Analyze structure


The Beginning


Principles of X-ray diffractionWhat is a

crystal?

•The unit cell is the basic building block of the crystal

•The unit cell can contain multiple copies of the same molecule whose positions are governed by symmetry rules


Proteins and crystallisation

•Proteins must be homogenous & monodispersed.

•Need large amount (mg quantities)

•Is it stable ( salt, pH, temp)

•Will modifications have to be made?

•What type of protein is it? Has anything similar been crystallized before?

•Proteins must be pure (> 99%) & fully folded Check the activity of your protein if you have an assay Check folding by other spectroscopic methods


•Crystallisation of proteins ‘controlled’ precipitation of the protein.

•Protein aggregates associate & form intermolecular contacts that resemble those found in the final crystal. Aggregates reach the critical nuclear size, growth proceeds by addition of molecules to the crystalline lattice.

•The processes of nucleation and crystal growth both occur in supersaturated solutions.

Precipitant

Cover-slip sealed with vacuum grease

Protein in “Hanging drop”

Process controlled by:

•Temp

•pH

•Salt conc

•Precipitants (PEG, ethanol)


Diffraction Apparatus


Synchrotron radiationMore intense X-rays at shorter wavelengths mean higher resolution & much quicker data collection


Experimental setup


Remove cover slip and fish out crystal with a small nylon loop

Mount loop on goniostat in a stream of nitrogen gas

Surface tension of the liquid in the loop holds crystal in place

Mounting crystals


Diffraction

•Each image represents the rotation of the crystal 1 degree in the X-ray beam.

•Each images gives us the position of each spot relative to all the others & there intensity.

•Intensity = square of amplitude.


Diffraction Principles

n= 2dsin


Diffraction Principles

A string of atoms CorrespondingDiffraction Pattern


The reciprocal lattice and the geometry of diffraction

X-ray source

X-ra

y

dete

ctor


Spacing between diffraction spots defines unit cell

1/a

1/b


Waves & the phase problem

The amplitudes of the diffracted X-rays can be experimentally measured, but the phases cannot = phase problem.

i.e. we don’t know the phase of each diffracted ray relative to the others!

X ?

A

Z

X

Y


The Phase Problem

• Diffraction data only records intensity, not phase information (half the information is missing)

• To reconstruct the image properly you need to have the phases (even approx.)– molecular replacement– direct methods– isomorphous replacement– anomolous dispersion


Structure factors & Fourier transforms

unit cell

F (h,k,l) = Vx=0 y=0 z=0 (x,y,z).exp[2I(hx + ky + lz)].dxdydz

A reflection electron density

All reflections phase

(x,y,z) = 1/V hkl F (h,k,l)exp[2I(hx + ky + lz) + i(h,k,l)

Electron density amplitude

At a point • The vector (amplitude and phase) representing the overall

scattering from a particular set of Bragg planes is termed the structure factor (F).

• Structure factors for various points on the crystal lattice correspond to the Fourier transform of electron density within the unit cell and vice-versa.


F T

Fourier Transform of a molecule


Fourier Transform of a

crystal


The Phase Problem

• Diffraction data only records intensity, not phase information (half the information is missing)

• To reconstruct the image properly you need to have the phases (even approx.)– molecular replacement– direct methods– isomorphous replacement– anomolous dispersion


Molecular replacement

• Requires a starting model for structure

• Can calculate back from structure to electron density to structure factors

• Works if model is 30 to 40 % identical to correct answer


Molecular Replacement

By determining the correct orientation and position of a molecule in the unit cell using a previously solved structure as a ‘search model’. This model can then be used to calculate phases


Isomorphous replacement (IR)

• Provides indirect estimates of the protein phase angles by observing the interference effects of the intensities on scattered beams by a heavy atom marker.

• All the electrons in the heavy atom will scatter essentially in the same phase.

• We can solve the positions of these heavy atoms because they are few in number and strong in signal.

• Using this estimate we can deduce the positions of the protein atoms and their phases


Anomalous scattering

• Scattering information of an atom whose absorption frequency is close to the wavelength of the source beam produces phase information

• Resolved anomalous scattering requires intensity measurements at one wavelength

• Multi-wavelength anomalous dispersion, requires intensity measurements at several wavelengths


•Using the structure factor calculation we can produce electron density maps for the whole protein.

•We then fit our protein model (co-ordinates X,Y,Z) inside the map.


Resolution

1.2 Å

2 Å

3 Å


Resolution

6Å: Outline of the model, feature such as helices can be identified.

3Å: Can trace polypeptide chain using sequence data, establish folding topology. Assign side chains.

2Å: Accurately establish mainchain conformation, assign sidechains without sequence data, I.d water molecules.

1.5Å : Individual atoms are almost resolved, detailed discription of water structure.

1.2Å: Hydrogen atoms may become visible.


Final Structure

But the work is not over yet!


Refinement• The process of building and rebuilding a model can cause

many errors in the structure.

1. Bond length,

2. Bond angle

3. Atomic clashes etc

• It is necessary to subject the structure to refinement in order to remove these errors and produce a better structure.

• Minimization

• Thermal parameters

• In order to further improve the model, it is refined using a simulated annealing protocol

• Refinement progress is monitored by following the agreement between the the observed data ( data collected) and the calculated data (data calculated from current model) = R factor


• R-factor The agreement between the the observed data (data collected) and the calculated data (data calculated from current model) the lower the number the better; typically around 20%

• Resolution The higher the resolution the more detail that can be seen 3.0Å is fairly low whilst 1.1Å is approaching atomic resolution

• B-factor Measure of thermal motion. i.e. how much energy each atom contains. Gives us information on mobility & stability

• Rms deviation Deviation of bond lengths & angles from ideal

Quality of the structure?


Deviation of bond lengths & angles from ideal. All based on the geometry of small molecules. Rms deviation for bond lengths should be less than 0.02Å and less than 4º for bond angles

Determined using a Ramachandran plot.

Rms deviation of bond length & bond angle


Absorption of Light


Protein chromophores:•Peptide bond•Amino acid side chains•Prosthetic groups

Peptide bond absorbance:•210 nm due to n transition•190 nm due to transition

Amino acid side chain absorbance:•Asp, Glu, Asn, Gln, His and Arg have transitions at the same wavelength where peptide absorbs

Protein concentration can be measured by measuring absorbance at 280 nm and by assuming that 1 mg ml-1 solution of protein has absorbance of 1.0

Absorption in the UV and visible range


Absorption and emission spectra of individual tryptophan residues, in the absence of energy transfer


Fourth derivative absorption spectrum

• Fourth derivatives of the absorption spectra have been documented as a valuable tool for studying structural changes in proteins.

• Protein fourth derivative spectra have been shown to be very sensitive to changes in the microenvironment (polarity, hydration, hydrophobic interactions, packing density) of tyrosine and tryptophan residues

Chauhan and Mande, Biochem J, 2001


Measurements of conformational properties using optical activity


Linearly polarised lightLinearly polarised light Right circular polarisationRight circular polarisation Left circular polarisationLeft circular polarisation


• Nearly all molecules of life are optically active

• There are four ways that an optically active sample can alter the properties of transmitted light: optical rotation, ellipticity, circular dichroism, circular birefringence

Linear Circular Elliptical


After passing through an optically active absorbing sample, the light is changed in two aspects:

1. The maximal amplitude E is no longer confined to a place, instead it traces an ellipse

Ellipticity = tan-1 (minor/major axis)

2. The orientation of the ellipse is an indication of optical activity. If the sample did not absorb any light, the ellipse would such small axial ratio that it would be equivalent to a plane-polarised light. In this case we will say that the plane polarised light has been rotated.

3. Orientation of the ellipse is the optical rotation. Optical rotation as a function of wavelength is called the optical rotatory dispersion (ORD).


Circular Dichroism


CD spectrum of a protein


Where can Circular Dichroism be used?


Measurements of conformational properties using fluorescence


Fluorescence• Chromophores are components of

molecules which absorb light• They are generally aromatic rings


FluorescenceE

NE

RG

Y

S0

S1

S2

T2

T1ABS FL I.C.

ABS - Absorbance S 0.1.2 - Singlet Electronic Energy LevelsFL - Fluorescence T 1,2 - Corresponding Triplet StatesI.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence

IsC

IsC

PH

[Vibrational sublevels]

Jablonski Diagram

Vibrational energy levelsRotational energy levelsElectronic energy levels

Singlet States Triplet States


Simplified Jablonski Diagram

S0

S’1En

erg

yS1

hvex hvem


Fluorescence

The longer the wavelength the lower the energy

The shorter the wavelength the higher the energyeg. UV light from sun causes the sunburn not the red visible light


Fluorescence Excitation Spectra

Intensity related to the probability of the event

Wavelengththe energy of the light absorbed or emitted


Corrected excitation spectra (corrected for source output and monochromator throughput) can be obtained by using a reference channel equipped with a "quantum counter". This is a concentrated dye solution (typically 3 mg/mL rhodamine B in ethylene glycol). A tiny fraction of the excitation beam is diverted to the reference detector. The quantum counter absorbs all of this light, and converts it (with 100% efficiency to fluorescence), the intensity of which is independent of wavelength between 220 and 580 nm. Any changes in lamp output or monochromator throughput will cause corresponding alterations in the output of the reference channel. By dividing the fluorescence signal by the reference signal, these wavelength-dependent variations are cancelled out. Unfortunately, the quantum counter will not entirely correct the emission spectrum. However, instrument manufacturers supply correction factors for their monochromators. Application of these will give an approximately correct spectrum. If more accuracy is needed, the spectrum of a known standard compound (fluorescing in the region of interest) can be compared to published standards.

j. Biological fluorophores 1) Intrinsic fluorophores a) Proteins Tryptophan dominates protein fluorescence spectra - high molar absorptivity - moderate quantum yield - ability to quench tyrosine and phenylalanine emission by energy transfer.

Free tyrosine has a relatively high fluorescent output, but is strongly quenched by trptophan in native proteins. Unless tyrosine and tryptophan are absent, emission from phenylalanine is not observed in protein fluorescent spectra.


Tryptophan is a good fluorophore

0

1000

2000

3000

4000

5000

6000

200 250 300 350 400 450 500

wavelength (nm)

ex

tin

cti

on

co

eff

icie

nt

0

50000

100000

150000

200000

250000

300000

flu

ore

sc

en

ce

em

iss

ion

absorption

fluorescence

note that this note that this fluorescence fluorescence expt used an expt used an excitation excitation of of 270nm270nm

we can consider solvent effects on its emission wavelength in the same way we did for absorption...

note that the note that the fluorescence fluorescence looks like a looks like a mirror image of mirror image of the 280nm the 280nm absorption absorption peak (and peak (and notnot the 220nm the 220nm peak)peak)


Absorption vs Emission for Trp

comparing our diagrams for absorption and emission – and assuming that protein interiors behave like organic solvents(!) – we predict:

Abs.Abs.

in waterin waterburiedburied in in proteinprotein

EEabsabs EEabsabs

proteiproteinn

waterwater

absorptiabsorptionon

EEemem EEemem

emissionemission

proteiproteinn

waterwater

Em.Em.


Effect of Ca2+ on Intrinsic Trp-fluorescence and on Fluorescence Anisotropy

▼ Wild type• Dome loop mutant

Blue shift and intensity enhancement upon addition of Ca2+

Change in anisotropy upon titration in the wild type, but not in the mutant


Raman Scatter• A molecule may undergo a vibrational

transition (not an electronic shift) at exactly the same time as scattering occurs

• This results in a photon emission of a photon differing in energy from the energy of the incident photon by the amount of the above energy - this is Raman scattering.

• The dominant effect in flow cytometry is the stretch of the O-H bonds of water. At 488 nm excitation488 nm excitation this would give emission at 575-595575-595 nm nm


Rayleigh Scatter• Molecules and very small

particles do not absorb, but scatter light in the visible region (same freq as excitation)

• Rayleigh scattering is directly proportional to the electric dipole and inversely proportional to the 4th power of the wavelength of the incident light

the sky looks blue because the gas molecules scatter more light at shorter (blue) rather than longer wavelengths (red)


Probes for Proteins

FITC 488 525PE 488 575APC 630 650PerCP™ 488 680Cascade Blue 360 450Coumerin-phalloidin 350 450Texas Red™ 610 630Tetramethylrhodamine-amines 550 575CY3 (indotrimethinecyanines) 540 575CY5 (indopentamethinecyanines) 640 670

Probe Excitation Emission


• Hoechst 33342 (AT rich) (uv) 346 460• DAPI (uv) 359 461• POPO-1 434 456• YOYO-1 491 509• Acridine Orange (RNA) 460 650• Acridine Orange (DNA) 502 536• Thiazole Orange (vis) 509 525• TOTO-1 514 533• Ethidium Bromide 526 604• PI (uv/vis) 536 620• 7-Aminoactinomycin D (7AAD) 555 655

Probes for Nucleic Acids


DNA Probes• AO

– Metachromatic dye• concentration dependent emission• double stranded NA - Green• single stranded NA - Red

• AT/GC binding dyes– AT rich: DAPI, Hoechst, quinacrine– GC rich: antibiotics bleomycin, chromamycin

A3, mithramycin, olivomycin, rhodamine 800


Probes for Ions

• INDO-1 Ex350Em405/480

• QUIN-2 Ex350 Em490

• Fluo-3 Ex488 Em525

• Fura -2 Ex330/360 Em510


pH Sensitive Indicators

• SNARF-1 488 575

• BCECF 488 525/620

440/488 525[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]

Probe Excitation Emission


Probes for Oxidation States

• DCFH-DA(H2O2) 488 525

• HE (O2-) 488 590

• DHR 123 (H2O2) 488 525

Probe Oxidant Excitation Emission

DCFH-DA - dichlorofluorescin diacetateHE - hydroethidineDHR-123 - dihydrorhodamine 123


Specific Organelle Probes

BODIPY Golgi 505 511

NBD Golgi 488 525

DPH Lipid 350 420

TMA-DPH Lipid 350 420

Rhodamine 123 Mitochondria 488 525

DiO Lipid 488 500

diI-Cn-(5) Lipid 550 565

diO-Cn-(3) Lipid 488 500

Probe Site Excitation Emission

BODIPY - borate-dipyrromethene complexesNBD - nitrobenzoxadiazoleDPH - diphenylhexatrieneTMA - trimethylammonium


Other Probes of Interest• GFP - Green Fluorescent Protein

– GFP is from the chemiluminescent jellyfish Aequorea victoria

– excitation maxima at 395 and 470 nm (quantum efficiency is 0.8) Peak emission at 509 nm

– contains a p-hydroxybenzylidene-imidazolone chromophore generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the primary sequence

– Major application is as a reporter gene for assay of promoter activity

– requires no added substrates


Energy transfer

excitationexcitation emissionemission

transfertransfer

A B

phycoerythrin-Texas Redphycoerythrin-Texas Red ECDECDphycoerythrin-cyanine5phycoerythrin-cyanine5 PC5PC5


Energy Transfer

• Effective between 10-100 Å only• Emission and excitation spectrum must significantly

overlap• Donor transfers non-radiatively to the acceptor

Inte

nsit

y

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2

Understanding biology through structures Course work 2006 Structure Determination and Analysis :...

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