This document summarizes several methods for determining protein structure: X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. X-ray crystallography involves growing protein crystals, exposing them to X-rays to generate diffraction patterns, and using the patterns to build 3D electron density maps of the protein. Nuclear magnetic resonance spectroscopy measures distances between atomic nuclei in soluble proteins by analyzing spectra from radiofrequency pulses applied in strong magnetic fields. Cryo-electron microscopy images frozen, hydrated protein samples with an electron microscope to determine large protein structures without the need for crystallization.

1. 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

2. Outline
Introduction
X-ray crystallography
Nuclear magnetic resonance
Cryo-Electron microscopy

3. 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

4. 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

5. Protein X-ray crystallography
o 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

6. 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.

7. http://pruffle.mit.edu/atomiccontrol/education/xray/xray_diff.php
o 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.

8. 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/spe
ctrpy/nmr/nmr1.htm

9. 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

10. 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

11. 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 film.
2.Vitreous section.
http://www.britannica.com/technology
/transmission-electron-microscope

12. 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.

13. References
1. http://www.britannica.com/technology/transmission-electron-microscope
2. http://www.particlesciences.com/news/technical-briefs/2009/protein-structure.html
3. https://archive.org/details/xrayscrystalstru00braguoft
4. http://www.theochem.unito.it/crystal_tuto/mssc2013_cd/tutorials/geometry/docpu/crystut.html
5. http://www.xtal.iqfr.csic.es/Cristalografia/parte_02-en.html
6. 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/mission
8. http://serc.carleton.edu/research_education/geochemsheets/techniques/SXD.html
9. https://science.education.nih.gov/supplements/nih4/technology/guide/info-technology.html
10. http://www.lsi.umich.edu/center-for-structural-biology/macromolecular-crystallization-and-
crystallography
11. http://www.rigaku.com/en/node/2449http://learn.crystallography.org.uk/education/how-do-we-study-
crystals
12. https://publications.nigms.nih.gov/structlife/chapter2.html
13. http://www.pnas.org/content/111/35/12769/F1.expansion.html
14. https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/nmr/nmr1.htm
15. http://chemistry.stackexchange.com/questions/42757/why-only-one-peak-is-observed-in-nmr-
spectrum-of-h2
16. http://www.ibet.pt/services/structural-biology-drug-discovery/protein-structure-and-drug-design

14. Thanks