protein structure determination

STRUCTURE DETERMINATION

Structure DeterminationAgenda

Cryo-Electron microscopy

Protein X-ray Crystallography

Structure Quality Measures

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Nuclear magnetic resonance (NMR)

Advantages & Disadvantages (X-Ray vs. NMR)

• Structure DeterminationVarious functions of biological system depend upon the

structure and function of proteins.

Determination of structure and functions of proteins assist

in scrutinizing the dynamics of proteins.

To understand the functions of proteins at a molecular

level, it is often necessary to determine their three-

dimensional structure.

Introduction

Introduction

Why Structure Determination ?

helps us in Understanding:

• How proteins interact with other molecules ?

• How they perform catalysis in the case of enzymes ?

• Interaction of protein with other molecules including

protein itself.

• Miscoding and/or misfolding of proteins associated with

diseases.

Introduction





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X-Ray Crystallography

• What is X-Ray Crystallography?

– A form of very high resolution microscopy.

– Enables us to visualize protein structures at the atomic level

– Enhances our understanding of protein function.

• What is the principle behind X-Ray Crystallography?

– It is based on the fact that X-rays are diffracted by crystals.

http://pruffle.mit.edu/atomiccontrol/education/xray/xray_diff_files/image002.gif

Why X-Rays? Not Others?

300 nm

10 nm

0.1 nm or 1 Å

Wavelength

Individual cells

and sub-cellular

organelles

Cellular

architecture

Shapes of large

protein molecules

Atomic detail of

protein

1.Light

1.Electron

1.X-Rays

VisualizationMicroscopy

Why use X-rays and crystals?Optical microscopy vs. X-ray diffraction

• X-rays is in the order of atom diameter and bond lengths, allowing these to be

individually resolved.

• No lenses available to focus X-rays. Crystal acts as a magnifier of the

scattering of X-rays.

http://classes.soe.ucsc.edu/bme220/Spring07/NOTES/Xraycryst.IMcNae_MWalkinshaw.pdf



• 1. Protein purification.

• 2. Protein crystallization.

• 3. Data collection.

• 4. Structure Solution (Phasing)

• 5. Structure determination (Model building and refinement)

Steps in Structure Determination

http://www2.uah.es/farmamol/New_Science_Press/nsp-protein-5.pdf


• What is Protein Purification?

– is a series of processes intended to isolate one or a few proteins from a

complex mixture, usually cells, tissues or whole organisms.

• Why Protein Purification?

– Characterization of the function.

– Structure

– Interactions of the protein.

• Requirements

– minimum of 5 to 10 milligrams pure soluble

– protein are required with better than 95% purity

Step1:Protein Purification




• Why Crystallization:

– X-ray scattering from a single unit would be unimaginably weak.

– A crystal arranges a huge number of molecules in the same orientation.

– Scattered waves add up in phase and increase Signal to a level which

can be measured.

– This is often the rate-limiting step in straightforward structure

determinations, especially for membrane proteins

Step2:Protein crystallization

http://xray.bmc.uu.se/~kaspars/xray.ppt



Crystals MUST be:

Small in size:

•Less than 1 millimeter

PERFECT:

•No cracks

•No Inclusions, such as air

bubbles

Improving Crystal Quality

Hanging Drop Method

Hanging Drop Method:

1 to 5μl protein solution is suspended over

a 1 ml reservoir containing precipitant

solution

e.g. ammonium sulfate solution or

polyethylene glycol



Mounting Crystals:• Crystals are mounted in a way so that the sample

can be rotated and an X‐Ray beam can be passed

through the sample.

• Methods of mounting include using either a capillary

or a tube.

• Both capillary and tubes are mounted on a

goniometer.


Step3:Data collection:

Exposing X‐Rays:Once the crystals are correctly mounted, they are

exposed to X‐Ray Beams. X‐Ray Sources include:

• Synchrotron: gives high resolution and luminosity

• X‐Ray generators: for smaller, laboratory use

http://serc.carleton.edu/research_educati

on/geochemsheets/techniques/SXD.html


• The source of the X-rays is often a synchrotron.

• The typical size for a crystal for data collection may be 0.3 x 0.3 x

0.1 mm.

• The crystals are bombarded with X-rays which are scattered from

the planes of the crystal lattice.

• The scattered X-rays are captured as a diffraction pattern on a

detector such as film or an electronic device.

Step3:Data collection:

http://pruffle.mit.edu/atomiccontrol/education/xray/xray_diff_files/image006.gif


• Rotate crystal through 1 degree and Record XRD pattern

• If XRD pattern is very crowded, reduce the degree of rotation

• Repeat until 30 degrees were obtained

• Sometimes 180 degrees depending on crystal symmetry

• Lower the symmetry= More data are required

• For high resolution, use Synchrotron

Step3:Data collection

http://upload.wikimedia.org/wikipedia/commons/d/de/Kappa_goniometer_animation.ogg


Step4:Structure Solution (Phasing)

A typical diffraction pattern from a

protein crystal

GOAL= From Diffraction Data to Electron Density

The 3D structure obtained above is

the electron density map of the

crystal.http://www.chem.ucla.edu/harding/IGOC/E/electron_density_map01http://www.chem.ucla.edu/harding/IGOC/D/diffraction_pattern01.jpg

Phasing

Purification & Crystalization

Diffraction Phasing

• What is the Phase problem?

– In the measurement of data from an X-ray crystallographic

experiment only the amplitude of the wave is determined.

– To compute a structure, the phase must also be known.

– Since it cannot be determined directly, it must be determined

indirectly or by some other experiment.



• Methods for solving the phase problem

– Molecular Replacement (MR)

– Multiple/Single Isomorphous replacement (MIR/SIR)

– Multiple/Single wavelength Anomalous Diffraction(MAD/SAD)

• Principle using Fourier Transform (FT) :

– FT of the diffraction data gives us a representation of the contents

of the crystal.






Step5: Structure determination (Fitting):

• Fitting of protein sequence in the electron density.

• Electron density – Not self explanatory

• Can be automated, if resolution is close to 2Å or better.

• What can be interpreted is largely defined by resolution.




Step5: Structure determination (Refinement):

Automated improvement of the model, so it explains the observed data

better.

The phases get improved as well, so the electron density maps get better.



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Introduction:

• The aim:Measure set of distances between atomic nuclei.

• Why?– For proteins that are hard to crystallize.

– For proteins that can be dissolved at high concentrations.

– To study dynamics of the protein: conformational equilibria,

folding and intra-, intermolecular interactions.

Nuclear magnetic resonance (NMR)The concept

• The base is the nucleus Spin.

• Spin is characterized by angular momentum vector.

• Can be parallel or anti-parallel external magnetic field.

• Forms energy states , low and high

• Applying radio frequency can change the states.

http://www.umkcradres.org/Spec/RADPAGE/Magnet2.jpg


• Perturbation of the spins causes a NMR signal to be observed.

• The signal consists of RF waves with frequencies that match the energy difference between the spin states of the individual nuclei involved.

• The resonance frequencies of different types of nuclei are widely different.

http://en.wikipedia.org/wiki/File:NMR_EPR.gif


• Chemical shift is the resonant frequency of a nucleus relative to a standard.

• Nuclear Overhauser effect (NOE) permits distance measurements between nuclei.

http://www.cs.duke.edu/brd/Teaching/Bio/asmb/current/2papers/Intro-reviews/flemming.pdf


• 1. Protein solution.

• 2. NMR spectroscopy (data collection)

• 3. Sequential resonance assignment

• 4. Collection of conformational constraints

• 5. Structure calculation

Steps in Structure Determination

http://uah.es/farmamol/New_Science_Press/nsp-protein-5.pdf

• Highly purified protein preparation.

• Unlike crystallography, structure determination by NMR is carried out on aqueous sample.

• Usually, the sample consists of between 300 and 600 microlitres with a protein concentration in the range 0.1 – 3 millimolar.

• The purified protein is usually dissolved in a buffer solution

Nuclear magnetic resonance (NMR)Step1: Protein solution

• Each distinct nucleus produces a chemical shift by which it can be recognized .

• Overlapping chemical shifts , So!

• Two main experiments categories

- One where magnetization is transferred through the chemical bonds.

- One where the transfer is through space.

Nuclear magnetic resonance (NMR)Step2: NMR spectroscopy (data collection)

• Map chemical shift to atom by sequential walking .

• Application of multidimensional NMR spectroscopy allowed the development of general strategies for the assignment .

• Take advantage of the known protein sequence.

Nuclear magnetic resonance (NMR)Step3: Sequential resonance assignment

http://en.wikipedia.org/wiki/File:1H_NMR_Ethanol_Coupling_shown.GIF

• Can be obtained within one week.

• The assignment of inter-atomic distances based on proton/proton NOEs observed in is quite time consuming.

• Structure calculation and NOE assignment is an iterative process.

Nuclear magnetic resonance (NMR)Step3: Sequential resonance assignment

• Geometric conformational information to be derived from the NMR data.

• Distance restraints.

• Restraints angle .

• Orientation restraints.

• Chemical shift data, provides information on the type of secondary structure

Nuclear magnetic resonance (NMR)•Step4: Collection of conformational constraints

• Determined restraints is the input.

• Using computer programs The process results in an ensemble of structures .


•Step5: Structure calculation

http://en.wikipedia.org/wiki/File:Ensemble_of_NMR_structures.jpg




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• Every experiment has associated errors

• Random errors will affect the reproducibility and precision of the

resulting structures

• Systematic errors affect the accuracy of the model

• Precision indicates the degree of reproducibility of the

measurement and is often expressed as the variance of the

measured data set under the same conditions

• Accuracy, however, indicates the degree to which a measurement

approaches its correct value

• Ideally, a model of a protein will be more accurate the more fit the

actual molecule it represents and will be more precise as there is

less uncertainty about the positions of its atoms


Definitions

• R-Factor

– A measure of agreement between the crystallographic model and the

original X-ray diffraction data.

– The R-factor is used to assess the progress of structure refinement, and

the final R-factor is one measure of model quality.

– The R-factor is calculated as follows:

• |Fobs| is derived from the measured intensity of a reflection in the

diffraction pattern

• |Fcalc| is the intensity of the same reflection calculated from the

current model

– The absolute range of values is 0 to 1, the lower the better structure

– Usually ranges between 0.6 and 0.2


X-Ray Crystallography Quality Assessment

• Free R-Factor

– The free R-factor, Rfree, is computed in the same manner as R-Factor,

but using only a small set of randomly chosen intensities (the "test set")

which are set aside from the beginning and not used during refinement

– They are used only in the cross-validation or quality control process of

assessing the agreement between calculated (from the model) and

observed data

• The quantities RSR, Rmerge and Rsymm are similarly used to describe

the internal agreement of measurements in a crystallographic data

set.

– These quantities are generally less used, and they are explained on our

Wiki


X-Ray Crystallography Quality Assessment

• Knowledge-based quality measures

– Knowledge-based (KB) metrics describe how well the structure model

conforms to expectations

– They use selected features, such as:

• Bond length and bond angle distributions, dihedral angle distributions,

atomic packing, hydrogen bond geometries, and other geometric features.

– Ideal values are derived high-resolution X-ray structures

• Model versus data measures

– The most general form of MvD validation involves comparison of

distances and dihedral angles in models with the corresponding

experimental restraints.

– MvD measures are used widely with NMR


NMR Quality Assessment

• Common MvD Measures

– Root-Mean Square Deviation (RMSD)

• A common approach to asses the quality of NMR structures and to

determine the relative difference between structures

• An rmsd is a measure of the distance separation between

equivalent atoms:

• Two identical structures will have an rmsd of 0Å

– RPF Quality Scores

• Recent efforts in NMR structure validation have included increased

use of RPF Scores to calculate the ‘‘goodness-of-fit’’ between the

3D protein NMR structures and experimental NOESY peak list



http://biomaps.rutgers.edu/JACS_127_1665_2005.pdf

http://biomaps.rutgers.edu/JACS_127_1665_2005.pdf

• RPF Quality Scores

– Recall

TP / (TP + FN)

– Precision

TP / (TP + FP)

– F-measure

• Overall performance score calculated from the recall and precision

• It provides measure of the overall fit between the query model

structure and the experimental data

(2 x Recall x Precision) / (Recall + Precision)







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X-Ray Pros X-Ray Cons NMR Pros NMR Cons

Get whole 3D structure

by analysis of good

crystallized material

Protein has to form

stable crystals that

diffract well

Can provide information

on dynamics and

identify individual side-

chain motion

Requires concentrated

solution - therefore

danger of aggregation

Produces a single

model that is easy to

visualize and interpret

Crystal production can

be difficult and time

consuming

Secondary structure can

be derived from limited

experimental data

Currently limited to

determination of

relatively small proteins

More mathematically

direct image

construction

Inability to examine

solutions and the

behavior of the

molecules in solution

Free from artifacts

resulting from

crystallization

A weaker interpretation

of the experimental

data

Quality indicators

available (resolution, R-

factor)

There is no chance for

direct determination of

secondary structures

Useful for protein-

folding studiesProduces an ensemble

of possible structures

rather than one model

Large molecules can be

determined

Unnatural, non-

physiological

environment

Closer to biological

conditions in some

respects

Advantages & Disadvantages

X-Ray vs. NMR






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Another method for structure determination

• Definition:

– is a new technology for studying the architecture of cells, viruses and

protein assemblies at molecular resolution.

• Biological specimens:

1. Thin film

2. Vitreous sections


Another method for structure determination

• Advantages :

1. Allows the observation of specimens that have not been stained or

fixed in any way

2. Showing them in their native environment

3. Less in functionally irrelevant conformational changes

• Disadvantages:

1. Expensive

2. The resolution of cryo-electron microscopy maps is not high enough


Questions





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THANK YOU!

Unit Cell vs. Biological Cell

• Unit Cell: Asymmetric unit is the smallest portion of a crystal

structure to which symmetry operations can be applied in order to

generate the complete unit cell (the crystal repeating unit)

• Biological Cell: macromolecular assembly that has either been

shown to be or is believed to be the functional form of the molecule.

hemoglobin

(αβ)2

Unit Cell vs. Biological Cell

• Thus, a biological assembly may be built from:

• one copy of the asymmetric unit

• a portion of the asymmetric unit

• Asymmetric unit with multiple biological assemblies


Step1:Protein Purification(Backup)

A figure summarizing the steps involved in a metal binding strategy for protein

purificationhttp://upload.wikimedia.org/wikipedia/commons/thumb/e/e9/Protein_Purification_MetalBinding.tif/lossy-page1-320px-Protein_Purification_MetalBinding.tif.jpg


Braggs law

Step2:Protein crystallization(Backup)

http://www.eserc.stonybrook.edu/ProjectJava/Bragg/

Scattered beams in phase,

they add up

Scattered beams not in

phase, they cancel each other

nl = 2d sinq

• The biological material is spread on an electron microscopy grid and is preserved in a frozen-

hydrated state by rapid freezing, usually in liquid ethane near liquid nitrogen temperature. By

maintaining specimens at liquid nitrogen temperature or colder, they can be introduced into the

high-vacuum of the electron microscope column. Most biological specimens are

extremely radiation sensitive, so they must be imaged with low-dose techniques (usefully, the low

temperature of cryo-electron microscopy provides an additional protective factor

against radiation damage).

• Consequently, the images are extremely noisy. For some biological systems it is possible to

average images to increase the signal-to-noise ratio and retrieve high-resolution information about

the specimen using the technique known as single particle analysis. This approach in general

requires that the things being averaged are identical, although some limited conformational

heterogeneity can now be studied (e.g. ribosome). Three-dimensional reconstructions from cryo-

EM images of protein complexes and viruses have been solved to sub-nanometer or near-atomic

resolution, allowing new insights into the structure and biology of these large assemblies.

• Analysis of ordered arrays of protein, such as 2-D crystals of transmembrane

proteins or helical arrays of proteins, also allows a kind of averaging which can provide high-

resolution information about the specimen. This technique is called electron crystallography.

Thin film

• The thin film method is limited to thin specimens (typically < 500 nm) because the electrons

cannot cross thicker samples without multiple scattering events. Thicker specimens can be

vitrified by plunge freezing (cryofixation) in ethane (up to tens of μm in thickness) or more

commonly by high pressure freezing (up to hundreds of μm). They can then be cut in thin sections

(40 to 200 nm thick) with a diamond knife in a cryo ultramicrotome at temperatures lower than -

135 °C (devitrification temperature). The sections are collected on an electron microscope grid

and are imaged in the same manner as specimen vitrified in thin film. This technique is called

cryo-electron microscopy of vitreous sections (CEMOVIS) or cryo-electron microscopy of frozen-

hydrated sections.

Vitreous sections

protein structure determination

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