Summary of methods for protein structure determination

1)El Sayed Sabry Mohamed 2)El Sayed Fawzy El Sayed 3)El Sayed Nader Qasem 4)Altahra El Sayed Shetewy

5) Amal Ashraf Abd Alaziz

Outline

Introduction

X-ray crystallography

Nuclear magnetic resonance

Cryo-Electron microscopy

Introduction

Protien structure determination:Determining 3D structure of protein help us to understand mechanism of action of protein and it’s functions.

http://www.particlesciences.com/news/technical-briefs/2009/protein-structure.html

Protein X-ray crystallography

o Molecule size is too small to see under light microscope as it’s wave length(400-700nm)is much larger than molecule size.

o we use x-ray radiation to look to molecules as it’s

wave length(0.1nm) is much smaller.

X-ray crystallography principle: -That X-rays are diffracted by crystal. -Direct detection of atom position in crystal .

http://www.lsi.umich.edu/center-for-structural-biology/macromolecular-crystallization-and-crystallography

Protein X-ray crystallographyo Steps: 1.Protein purification: Minimum 5 to 10 mg pure soluble protein are

required with better than 95% purity. 2.Protein crystallization: 1. Disorder of unit cell. 2. Vibration of molecule. 3. Distortion in crystallization.

http://cbs.umn.edu/nanoliter-crystallization-facility/mission

Protein X-ray crystallography

crystallization –Hanging Drop Method: 1 to 5μl protein solution is suspended over 1 ml reservoir containing precipitant solution.

3.Date collection: -Mounting crystal…

-Crystals mounting by rotating them and X-ray

beams passed through them.

-This method include using a capillary or tube.

-Both capillary and tube mounted in goniometer.

http://pruffle.mit.edu/atomiccontrol/education/xray/xray_diff.phpo The X-ray source is often synchrotron(has high resolution).o The typical size of crystal is(0.3x0.3x0.1nm).o X-rayes penetrate crystal ,scattered and captured as diffraction pattern on a detector.o Rotate crystal and repeat the process in different degree until (30degree or180degree)and record XRDs pattern which Used to form electron density map of crystal.o This produced electron map may has some phasing problem can be treated by:

1. Molecular replacement. 2. Isomorphous replacement.

3. Fourier transform. Fitting protein sequence to get better electron map.

Nuclear magnetic resonance(NMR)o The aim: Measure set of distance between atomic nuclei.• Used for protein that are hard to crystallize or can be dissolved at high concentration.• NMR principle: -Based on nucleus spin( have angular momentum vector). -Spin can be parallel,anti parallel external magnetic field(forms energy state(low, high)). -Applying radiofrequency change this state.

https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/nmr/nmr1.htm

Nuclear magnetic resonance(NMR)o Steps: 1.Protein solution: Highly purified protein solution(300-600µl with protein conc.(0.1-3ml.M). 2.Data collection: Distinct nucleus produce chemical shift in two main experiments category:

-One where magnetization is transferred through the chemical bonds. -One where the transfer is through space.

3.Sequential resonance assignment: - Map chemical shift to atom by sequential walking. - Take the advantage of the known protein sequence. - The assignment based on proton/proton NOEs observed in is quite time consuming.

http://chemistry.stackexchange.com/questions/42757/why-only-one-peak-is-observed-in-nmr-spectrum-of-h2

Nuclear magnetic resonance(NMR)4.Collection of conformational constraints: Geometric conformational information derived from NMR.. 1.Distance between nuclei. 2.Angles between bonds. 3.Motion in solution. Chemical shift date provide information on the type of 2ry structure.5.Structure calculation: -Determined restraints is the input which used by computer programs -This process give us ensemble of structure.

http://www.ibet.pt/services/structural-biology-drug-discovery/protein-structure-and-drug-design

Cryo-Electron microscopy

o It is a new technology for studying the architecture of cells, viruses and protein assemblies at molecular resolution.

• Principle of Cryo-EM When a beam of electrons is passed through specimen a part of it is transmitted and this part when projected on fluorescent screen its image can be seen by the observer.

o Biological specimen: 1.Thin fi lm. 2.Vitreous section.

http://www.britannica.com/technology/transmission-electron-

microscope

Cryo-Electron microscopy

Advantages Disadvantages

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.

1. Expensive.2. The resolution of cryo-

electron microscopy maps is not high enough.

References1. http://www.britannica.com/technology/transmission-electron-microscope2. http://www.particlesciences.com/news/technical-briefs/2009/protein-structure.html3. https://archive.org/details/xrayscrystalstru00braguoft4. http://www.theochem.unito.it/crystal_tuto/mssc2013_cd/tutorials/geometry/docpu/crystut.html5. http://www.xtal.iqfr.csic.es/Cristalografia/parte_02-en.html6. https://www.thermofisher.com/eg/en/home/life-science/protein-biology/protein-biology-learning-

center/protein-biology-resource-library/pierce-protein-methods/overview-crosslinking-protein-modification.html1

7. http://cbs.umn.edu/nanoliter-crystallization-facility/mission8. http://serc.carleton.edu/research_education/geochemsheets/techniques/SXD.html9. https://science.education.nih.gov/supplements/nih4/technology/guide/info-technology.html10. http://www.lsi.umich.edu/center-for-structural-biology/macromolecular-crystallization-and-

crystallography11. http://www.rigaku.com/en/node/2449http://learn.crystallography.org.uk/education/how-do-we-study-

crystals12. https://publications.nigms.nih.gov/structlife/chapter2.html13. http://www.pnas.org/content/111/35/12769/F1.expansion.html14. https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/nmr/nmr1.htm15. http://chemistry.stackexchange.com/questions/42757/why-only-one-peak-is-observed-in-nmr-

spectrum-of-h216. http://www.ibet.pt/services/structural-biology-drug-discovery/protein-structure-and-drug-design

Thanks