![Page 1: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/1.jpg)
MOLECULAR BIOTECHNOLOGY
Design and Synthesis of Bioactive Compounds
Luigi Vitagliano
Istituto di Biostrutture e BioimmaginiConsiglio Nazionale delle RicercheVia Mezzocannone 16, 80134 Napoli
![Page 2: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/2.jpg)
MOLECULAR BIOTECHNOLOGY
Design and Synthesis of Bioactive Compounds
Lecturer : Luigi Vitagliano
Phone: 0812534506
E-mail: [email protected]
Slides of the lectures can be downloaded from the page
Additional material for further reading will be provided
throughout the course
![Page 3: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/3.jpg)
Synthetic program of the course
Structural Biology
The state of the art
Experimental approaches
X-ray crystallography
Electron microscopy
Computational approaches
Protein structure prediction methods
Molecular dynamics simulation
Structure validation
Successful examples: The ribosome machinery and the DNA structure
Rational Design of bioactive molecules
Properties of protein structures
Ligand-protein interactions
Structure-based drug discovery
Ligand-based drug discovery
Fragment-based drug discoveryClassical and recent successful examples of rational drug design
![Page 4: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/4.jpg)
Lesson 1
Part a
Structural Biology
• The state of the art
• Experimental approaches
• X-ray crystallography
![Page 5: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/5.jpg)
Overview of the Structure-based drug discovery
A key factor in the success of this strategy is the accurate structural
characterization of the target that is frequently a protein or a nucleic acid
![Page 6: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/6.jpg)
Structure-based drug designi
![Page 7: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/7.jpg)
Biomolecoles
‘Small’ molecoles: Water, Vitamines, Hormons, Lipids, Metabolites…
Macromolecules: Proteins, Nuclecic acids (DNA/RNA), Polysaccharides
Molecules found in living organisms
HemoglobinC2952H4664O832N812S8Fe4
Proteins are characterized by a remarkable structural complexity being made of thousands of atoms
WaterH2O
GlucoseC6H12O6
![Page 8: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/8.jpg)
Complexity of Nucleic acids: DNA and RNAChains of DNA are made of millions of nucleotides
Current record for sequence deconding is of 2.3 million bases of human DNA in one step
Formula Cxxxxxxxx Oxxxxxxx Hxxxxxxxx Pxxxxxx Nxxxxxxxx
![Page 9: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/9.jpg)
Chimie dans l’espace - Discovery and importance of
stereochemistry (1875)
The definition of the three-
dimensional structure is crucial for
macromolecules since they may
adopt, in principle, an enormous
number of distinct states
![Page 10: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/10.jpg)
Structure – function relationships in water
H2O
The central oxygen atom binds two hydrogen atoms
and has two lone pairs of electrons
VESPR Rules
Bent structure
Negative pole
Positive pole
![Page 11: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/11.jpg)
Structure – function relationships in water
+
- +
+ -
-
+
+ - +
+ --
+ - +
+ ---
-
Ionic crystal
+
-
Hydration
![Page 12: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/12.jpg)
A biochemical/biological problem
Identification of the key molecular players
Preparation of the samples for the structural investigation
Experiment(s)
Analysis of the biomolecule structure
If everything works, you end up with ……
…..many additional biochemical/biological problems
The structural biological approach
![Page 13: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/13.jpg)
Protein Chemistry
Linear polypeptide chain
N Nitrogen
O Oxygen
C Carbon
H Hydrogen
R Side Chain
![Page 14: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/14.jpg)
Protein three-dimensional structure
![Page 15: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/15.jpg)
Structure - Function relationship
![Page 16: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/16.jpg)
Fibrous proteins These proteins are characterized by very repetitive sequence motif (examples Collagen, wool,
silk…) - They have frequently a structural (mechanical) role – They share significant analogies
with synthetic polymers.
Globular proteins These are characterized by a rough globular shape and presents a large variability in
aminoacid composition and sequence.
Intrinsically disordered proteins (IDP)These are proteins that do not present an intrinsic well-defined three-dimensional structure in
their biological active state. They tend to become structured upon interaction with other
biological partners. They often have a low complexity sequence (repetitions of specific
aminoacid traits).
All type of proteins play crucial role in biological processes. From a chronological point of view
fibrous proteins have been the first to be structurally characterized. They were followed by
globular proteins, that for their huge diversity and complexity are far to be fully characterized.
IDPs represent an emerging subject of the scientific community.
Proteins are often classified according to their structural properties
![Page 17: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/17.jpg)
The first structural characterization of a globular protein was accomplished by
Kendrew (Myoglobin 1958)
The aspect of Kendrew’s model of myoglobin was a horrible, visceral-looking
object, since he had used a long sausage of plasticine
What about the structure of globular proteins?
Upon seeing the structure of myoglobin (Fig. 1) at 6 Å resolution, Kendrew et al. said, “Perhaps the most remarkable features of the molecule are its complexity and its lack of symmetry. The arrangement seems to be almost totally lacking in the kind of regularities which one instinctively anticipates, and it is more complicated than has been predicated by any theory of protein structure. Though the detailed principles of construction do not yet emerge, we may hope that they will do so at a later stage of the analysis.”
![Page 18: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/18.jpg)
Enchanting protein shapes of globular proteins
![Page 19: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/19.jpg)
90°
Scaring protein shapes
Resuscitation promoting factor B da M. tuberculosis
.
![Page 20: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/20.jpg)
A classical exampleHemoglobin Sickle Cell Anaemia
This is genetically tramistted since in heterozygotes the mutation producese resistenceagainst malaria The mutated gene is wdespreadin Africa
Due to its high concentration mutated Hb may form fibers
A single residue mutation may cause a disease
Mutation : Glu Val
In erythrocytesHemoglobin isabundant: 340 mg/ml
Deoxy form
![Page 21: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/21.jpg)
Normal and mutated Hb
![Page 22: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/22.jpg)
Fructose intoleranceMutations in Aldolase B
Incidence 1/20000.
It is fundamental the elimination of fructose from diet
A single residue mutation may cause a disease
![Page 23: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/23.jpg)
Misfolded proteins
Globular proteins in specific conditions may aggregate to form insoluble species Amyloid fibers
Beta-rich structures
![Page 24: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/24.jpg)
Misfolding of proteins ‘
Trasmissible encelopathiesCow
Human Disease: vCJD variant of the diseaseCreutzfeldt-Jakob (nota dal 1996)
![Page 25: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/25.jpg)
Determination of the three-dimensional structure of
biological macromolecules
The status of the art
![Page 26: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/26.jpg)
1 Å 1 nm 10 nm 1 mm 10 mm 0.1 mm100 nm
NMR
X-ray crystallography
AFM
single particle Cryo-EM
Electron tomography
Light microscopy
atoms proteins viruses bacteria cells
Techniques generally used for the structural
characterization of Bio-macromolecules
![Page 27: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/27.jpg)
The paradigm of function-structure relationships
Structure - Function
Experiments
X – Ray crystallography
NMR Nuclear Magnetic resonance
Cryo-EM Electron Microscopy
‘Modeling’
Molecular dynamics
‘Docking’
Computations
![Page 28: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/28.jpg)
Number of protein structures reported in the
Protein Data Bank
The vast majority of these structures (85%) has been so far
determined by X-ray crystallography
![Page 29: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/29.jpg)
Some classical triumps of x-ray crystallography
a) Ribosome
b) Rhodopsin
c) Reovirus core particle
d) HMG-CoA reductase
e) RNA polymerase II
f) ATP synthase
g) Nucleosome core particle
h) Mismatch repairprotein
g) HIV envelope glycoprotein
gp120
![Page 30: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/30.jpg)
Not only large macromolecules……..
Examples of recent fundamental peptide structural studies
Amyloid-forming peptides (4 to 7 residues long)These structures became models for fibers of proteins involved in neurodegenerative diseases
X-ray structures
David Eisenberg group
Nelson et al. 2005, Nature
Sawaya et al 2007 Nature
Wiltzius et al. 2009 Nature Structure and Molecular biology
Sawaya et al. 2009 PNAS
Apostol et al. 2010 JBC
![Page 31: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/31.jpg)
X-ray crystallography and proteins: a happy marriageSince 1901 26 Nobel prizes have been awarded to 43 sceintists in disciplines
related to crystallography. A significant portion of these were for the characterization of large biomolecules
Recent Nobel prizes awarded to studies conducted using X-ray diffraction
2012 Chemistry
Lefkowitz e Kobilka –G-coupled receptors- Protein Crystallography
2011 Chemistry
Shechtman – Discovery of quasicristalls– Basic crystallography
2010 Physics
Geim e Novoselov – Graphene discovery and characterization – Materials crystallography
2009 Chemistry
Ramakrishnan, Steitz e Yonath – Ribosome structure– Protein Crystallography
2006 Chemistry
Kornberg – Tracription factors– Protein Crystallography
2003 Chemistry
MacKinnon – Potassium channels– Protein Crystallography
2017 CrioElectron Microscopy, 2013 Computational biology - 2002 NMR of proteins
![Page 32: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/32.jpg)
![Page 33: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/33.jpg)
This scenario is going to change soon….The coming era of cryo-electron microscopy
![Page 34: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/34.jpg)
After fifty years (and thousands of publications)
the mecahnism is still debated, Sept 2013
Protein funtionality: not just structure
Importance of protein dynamics
How does haemoglobin work?
![Page 35: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/35.jpg)
Determination of the three-dimensional structure of
biological macromolecules by X-ray diffraction
techniques
![Page 36: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/36.jpg)
Basic experiment
Main components
X-ray
Sample
Detector
![Page 37: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/37.jpg)
Proprieties of the X-rays
Electromagnetic radiation with wavelength
between 0.1 and 1000 Å
![Page 38: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/38.jpg)
Conventional X-ray generator
![Page 39: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/39.jpg)
X-ray sources for structural studies on proteins
Conventional
Synchrotron
![Page 40: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/40.jpg)
Syncrothron are devices in which charged particles (electron or
positron) circulate with speed close to that of light
The acceleration of these particles generates radiations
Components of a synchrotron
Bending magnets
![Page 41: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/41.jpg)
Focus
Diffraction
Image
Scheme of diffraction and imaging
Object Object
Detector
Lens
Diffraction
Diffraction
pattern
![Page 42: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/42.jpg)
Diffraction
Single emitter
![Page 43: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/43.jpg)
Constructive
interference
Destructive
interference
Diffraction & Interference
Diffusion from a multiple source
![Page 44: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/44.jpg)
Diffusion from disordered scatters
![Page 45: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/45.jpg)
![Page 46: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/46.jpg)
Diffusion from ordered scatters
![Page 47: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/47.jpg)
A diffraction image
![Page 48: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/48.jpg)
Take home messages
Scatters in these experiments are electrons – therefore we get
direct information on them
Sharp diffraction is a result of the ordered organization of the
electrons in the sample
All electrons of our sample contribute to the scattering in each
direction – each spot on the diffraction diagram depends on the
location of all electrons
Crystallography versus Spectroscopy
![Page 49: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/49.jpg)
Single crystal diffraction For systems with well defined shapes that could generate ordered
crystals - organic molecules, oligonucleotides and globular proteins
Fiber diffraction analysesFibrous systems
X-ray diffraction analyses
![Page 50: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/50.jpg)
Three-dimensional crystals
![Page 51: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/51.jpg)
A diffraction image
![Page 52: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/52.jpg)
In fibers the molecular entities are ordered along the so-called
fiber axis
![Page 53: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/53.jpg)
Fiber X-ray diffraction analysis
Theory
Fiber diffraction
![Page 54: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/54.jpg)
The discovery of the alpha-helix (1951)
The fiber diffraction
pattern
The 3D model
by Pauling and Corey
Wool diffraction pattern:
meridional reflection (5.1 Å)
equatorial reflection (10 Å)
![Page 55: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/55.jpg)
Successful modelling of other biomacromolecules came using
the same approach
Beta-sheet Pauling and Corey, 1951
Collagen Ramachandran and
Triple Helix Kartha 1954
Rich and Crick, 1961
![Page 56: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/56.jpg)
The most famous achievement of X-ray fiber diffraction
analysis
The DNA structure
![Page 57: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/57.jpg)
analisys
Single Crystal X-ray diffraction analysis
Data Collection
Data Analysis
![Page 58: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/58.jpg)
PROTEIN
CRYSTALLOGRAPHY
Main steps
![Page 59: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/59.jpg)
Single crystal X-ray crystallography:
The experimental setup
![Page 60: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/60.jpg)
Example of protein crystals
![Page 61: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/61.jpg)
X-ray sources for protein crystallography
Synchrotron
Conventional
![Page 62: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/62.jpg)
Crystallization
Phase transition: liquid - solid
Aqueous solution
of a protein
Amorphous solid
Crystal
Isotropic solid characterized by an
irregular organization of the
atoms/molecules (for example glass)
Homogeneous and anisotropic solid
characterized by a regular organization
of the atoms/molecules in the space
![Page 63: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/63.jpg)
Crystals are characterized by the repetition in the
three-dimensional space of a specific volume
that is defined as unit cell
a
b
c
Unit cell
The repeating structural motif may be:
an ion, an atom, a molecule or an ensemble
of molecules.
Three-dimensional lattice
![Page 64: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/64.jpg)
The seven crystal classes
The unit cell is defined by three
axes a, b, c and three angles.
Different combinations are
possible
![Page 65: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/65.jpg)
Examples of protein crystals
Due to their internal symmetry crystal are solids with planar
faces as polyhedra
![Page 66: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/66.jpg)
Main features of protein crystals
• High solvent (water) content (20-80%)
• Large solvent channels – the interactions between
mates in the crystals are weak
• Limited level of order – Limited size of protein crystals
• High fragility and sensitioty to external conditions
[Linear dimensions 0.1-1.0 mm (= 1015-1020 molecules);
Inorganic crystals may be seen at macroscopic level (for example quarz)]
[Salt bridges, hydrogen bonds, van der Waals interactions – a small fraction
of the protein atoms are involved in packing contacts]
[pH variations, temperature, ionic strength…]
![Page 67: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/67.jpg)
Crystal packing:
Elongation factor G
Solvent channels
• the protein may
be active
• it is possible to
diffuse small
molecules in the
crystals as ligands
and inhibitors
(soaking)
Ca trace
![Page 68: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/68.jpg)
Needs for crystallization
• Purity = absence of contaminants
• Homogeneity = absence of conformational (inter-
domain flexibility, equilibrium between conformers, aggregation, partial
denaturation…) and/or sequence (fragmentation, uncomplete
post-translational modifications… ) heterogeneity
• Quantity
• Biological activity
![Page 69: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/69.jpg)
• Purity
Effects of contaminants:
They may prevent crystal formation
They may perturb the crystal growth
They may favor the growth of disordered crystals
SDS gel: occurrence and
oimension of peptide/protein
contaminants
Needs for crystallization
![Page 70: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/70.jpg)
How can crystallization may be induced?
Phase transition: liquid - solid
Solubility (cs): maximum solute concentration at a given
temperature
Sovrasaturation (cp/cs) non-equilibrium; the concentration
of the solute (cp) is larger than its solubility (cs)
solubility
Solubility curve = solid-liquid
equilibrium
Co
nc. p
rote
in
Conc. precipitant
(A parameter that decreases
the protein solubility:
temperatura, pH, salt, …)
Phase Diagramprecipitation
sovrasaturation
![Page 71: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/71.jpg)
1) Nucleation: formation of stable aggragates high
sovrasaturation
2) Growth It may occur in the nucleation zone but is more
favore in the metastable region
3) End of the growth
Studies carried out on lisozyme
indicate that critical aggregates
are made of 30-40 molecules.
![Page 72: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/72.jpg)
Main chemico-physical parameters that affect
protein solubility
• Protein concentration
• Concentration and nature of the precipitating agent :
Salts: protein dehydration
Organic solvent : for example alcohols may reduce the
dielectric constant
Organic polymers (PEG, poliamynes)
• Temperature
• pH of the buffer
• Ionic strength
![Page 73: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/73.jpg)
Vapour diffusion techniques(Hanging - Sitting Drop)
Closed system: [ drop of (protein + precipitants) + precipitants+ air] it
evolves toward the equilibrium through the evaporation of the water
from the drop to the reservoir
Hanging Sitting
Examples of experimental settings
Soluzione
madre
![Page 74: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/74.jpg)
Factors that (may) affect protein crystallization
• Nature and concentration of the precipitant
• Protein concentration
• Buffer and pH
• Temperature
• Ionic strength
• Purity and homogeneity of the sample
• Additives, effettors, ligands
• Source of the macromolecule (organism, recom.)
• Redox status of the environement
• Ions
• Detergents
• Gravity, convection, sedimentation
![Page 75: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/75.jpg)
Protein crystallization faclities
RobotsCrystallization under microgravity
conditions
![Page 76: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/76.jpg)
“Beuty is not enough”
What is important is the diffraction pattern!
![Page 77: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/77.jpg)
A real diffraction image
![Page 78: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/78.jpg)
A more real case – resolution is limited
![Page 79: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/79.jpg)
Diffraction spot magnification
![Page 80: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/80.jpg)
Intensities of the pixels that constitute the spot
![Page 81: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/81.jpg)
Integration of the spot intensity
![Page 82: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/82.jpg)
Measure of the spot intensities
These intensities are proportional to the square of the
module of the structure factore F(hkl)
The structure factor is linked to the electron density
through an operation called Fourier
h k l
lzkykxi2explkhFV
1zvxρ
![Page 83: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/83.jpg)
Protein crystallography: basic concepts
![Page 84: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/84.jpg)
Electron density
map
model
fitting
![Page 85: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/85.jpg)
Model building in the elctron density maps
Main chain
Side chains
![Page 86: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/86.jpg)
Risolution structural details
3.5Å 4Å
bassa risoluzione
6Å: Solo caratteristiche grossolane
del modello sono rintracciabili. Ad
es. a-eliche.
3Å: Polypeptide chain trace
2Å: Backbone conformation
1.5Å : Individual atoms in the maps.
Detailed description of the solven
< 1.2Å: Hydrogen atoms (may)
become visible. 1.0Å altissima risol. 2.5Å media risol.
![Page 87: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/87.jpg)
Atomic resolution: better than 1.2 Å
“ultra-high resolution”: RNase A 0.87 Å
Individual atoms
Double conformations
Hydrogen atoms
JMB, 297, 713-732, 2000
![Page 88: MOLECULAR BIOTECHNOLOGY Design and Synthesis of …wpage.unina.it/luigi.vitagliano/Vitagliano_MolDes_L1a.pdf · MOLECULAR BIOTECHNOLOGY Design and Synthesis of Bioactive Compounds](https://reader036.vdocuments.site/reader036/viewer/2022063001/5f1564ad8aaf3a42310b97c1/html5/thumbnails/88.jpg)
ATOM 1 N VAL A 1 10.720 19.523 6.163 1.00 21.36
ATOM 2 CA VAL A 1 10.228 20.761 6.807 1.00 24.26
ATOM 3 C VAL A 1 8.705 20.714 6.878 1.00 18.62
ATOM 5 CB VAL A 1 10.602 22.000 5.966 1.00 27.19
ATOM 6 CG1 VAL A 1 10.307 23.296 6.700 1.00 31.86
ATOM 7 CG2 VAL A 1 12.065 21.951 5.544 1.00 31.74
ATOM 8 N LEU A 2 8.091 21.453 7.775 1.00 16.19
ATOM 9 CA LEU A 2 6.624 21.451 7.763 1.00 17.31
ATOM 10 C LEU A 2 6.176 22.578 6.821 1.00 18.55
ATOM 11 O LEU A 2 6.567 23.730 7.022 1.00 18.72
ATOM 12 CB LEU A 2 6.020 21.707 9.129 1.00 18.34
ATOM 13 CG LEU A 2 6.386 20.649 10.198 1.00 17.39
ATOM 14 CD1 LEU A 2 5.998 21.119 11.577 1.00 17.99
ATOM 15 CD2 LEU A 2 5.730 19.337 9.795 1.00 16.96
ATOM 16 N SER A 3 5.380 22.237 5.852 1.00 15.02
ATOM 17 CA SER A 3 4.831 23.237 4.928 1.00 16.59
ATOM 18 C SER A 3 3.725 24.027 5.568 1.00 14.84
ATOM 19 O SER A 3 3.095 23.717 6.591 1.00 14.40…
X Y Z
Coordinates Occup. B-factorAtom
Residue
A typical PDB file