Estructura de Proteínas
Métodos experimentales para la determinación de la estructura 3D
de proteínas
Métodos para la determinación de la estructura 3D de proteínas:
• Métodos experimentales– Cristalografía por rayos-X– Resonancia Magnética Nuclear (NMR)– Dicroísmo circular
• Métodos teóricos
Qué es la luz y cómo interactúa con la materia?
R-X
TEORÍA DE LA MATERIA
-PARTÍCULA (~m)
-MOLÉCULA (~A)
-ÁTOMO (~A)
-PROTÓN, NEUTRÓN, ELECTRÓN (fm)
-QUARK (am)
MODELO ATÓMICO DE BOHR
Werner Heisenberg Erwin Schrodinger
LA MECÁNICA CUÁNTICA Y LA NUEVA TEORÍA DEL ÁTOMO
LAS MOLÉCULAS PRESENTAN NIVELES DE ENERGÍA MÁS COMPLEJOS
ENERGÍA INTERMOLECULAR DEPENDIENTE DE LA DISTANCIA
LOUIS DE BROGLIE
LA MATERIA TIENE UNA NATURALEZA ‘DUAL’… A VECES SE COMPORTA COMO
PARTÍCULA, PERO OTRAS VECES SE COMPORTA COMO ONDA…
HIPÓTESIS DE DE’BROGLIE
h/mv
EXPERIMENTO DE DOBLE RENDIJA CON ELECTRONES
QUE ES LA MATERIA, PARTÍCULA U ONDA?
LAS DOS COSAS!
LA MATERIA TIENE UN COMPORTAMIENTO DUAL. A VECES SE
MANIFIESTA COMO PARTÍCULA, Y OTRAS VECES SE MANIFIESTA COMO
ONDA!
LA RADIACIÓN ELECTROMAGNÉTICA
-TEORÍA ONDULATORIA
-TEORÍA CORPUSCULAR
UNA ONDA ES LA PERTURBACIÓN DE UN CAMPO…
ONDAS LONGITUDINALES Y TRANSVERSALES
EN UNA ONDA NO HAY TRANSPORTE DE MATERIA, PERO SI SE TRANSPORTA ENERGÍA
A wave is a disturbance of a medium which transports energy through the medium without permanently transporting matter. In a wave, particles of the medium are temporily displaced and then return to their original position.
EL SONIDO ES UNA ONDA LONGITUDINAL
LA LUZ ES UNA ONDA ELECTROMAGNÉTICA
C =
REFLEXIÓN, REFRACCIÓN Y DIFRACCIÓN DE LA LUZ
POLARIZACIÓN DE LA LUZ
ESPECTRO DE LA RADIACIÓN ELECTROMAGNÉTICA
FUENTES DE LA RADIACIÓN E.M.
LA INTENSIDAD DE LA LUZ DEPENDE DEL NÚMERO DE FOTONES POR UNIDAD DE
TIEMPO POR AREA
LAS ONDAS ELECTROMAGNÉTICAS (FOTONES), PUEDEN PROPAGARSE EN
UN MEDIO MATERIAL ASÍ COMO EN EL VACÍO
CON LA ABSORCIÓN DE LA LUZ OCURRE UNA EXITACIÓN ELECTRÓNICA
ABSORCIÓN DE LA LUZ
LA MATERIA PUEDE SER ‘TRANSPARENTE’ A LA RADIACIÓN
Cómo explica Usted este fenómeno ?
Purified Protein
Solve the Phase Problem-MIR, MAD or MR (next class)
Proteins Can Form an Ordered Lattice
Celda unitaria tridimensional
Overview of X-ray Experiment
Each reflection has an hkl index
and a measured intensity
8 reflections selected from a 30,000 reflection data set shown to the right
2 0 3 1483.63 -1 -3 19999.9 3 -1 -2 6729.63 -1 -1 30067.13 -1 1 8227.03 -1 2 29901.53 -1 3 24487.53 -1 4 502.1
h k l I
X-Ray Crystallography
incoming x-ray
detector
diffractedx-ray
protein crystal
diffraction patterngoniometer controls crystal orientation
Crystal lattice
“Real Space”
Reflections
“Reciprocal Space”
Periodic
(x, y, z)
Discrete, complex
F (h, k, l)
I (h, k, l) (h, k, l)
Fourier transform - FT
intensity phase
X-ray Equipment in Delaware
RU-H3RX-ray generator
Area Detector
Close-up of Cryo Crystal
Patrón de difracción
Protein Crystal X-ray Diffraction
Zoom into a single reflection
The Diffraction Condition
Reflections are the result of constructive interference
Geometry determines the constructive condition
Bragg’s Law: n = 2d sin
sin = AB / d
d sin = AB
= 2d sin or
n = 2d sin
Fase de la onda y la nterferencia constructiva/destructiva
Each Reflection Index Defines a Set of Parallel Planes that Slice Through the Crystal
Miller indices hkl Reciprocal
Space
Real
Space
h a
k b
l c
Example:draw parallel planesthat define the single reflection hkl 2 3 4
Reciprocal space has all reflections out to diffraction limit
h 0 to 30k 0 to 35 just a hypothetical examplel 0 to 43
Crystallography and Crystallization
• To obtain X-ray diffraction data:– First, need to grow a crystal – Field of macromolecular crystallization
• Large parameter space (22 or so)
• Effects change over time
• Little theory
• Protein purification costly
• Tedious experimentation
Cristalización
Vapor Diffusion Method
Experimentation to grow protein crystals
Trial-and-errorExperimentation
Partial Success
Failure
Trial 1Trial 1
Trial 2Trial 2
Trial 3Trial 3
Success
Control Parameters
Observables
3. CrystalsWhat defines a crystal?Atoms, lattice points, symmetry, space groupsDiffractionB-factorsR-factorsResolutionRefinementModeling!
CrystalsWhat defines a crystal?
3D periodicity: anything (atom/molecule/void) presentat some point in space, repeats at regular intervals,in three dimensions.
X-rays ‘see’ electrons (r) = (r+X)
(r): electron density at position rX: n1a + n2b + n3cn1, n2, n3: integersa, b, c: vectors
Crystals
What defines a crystal?
crystalprimary building block:
the unit celllattice:
set of points withidentical environment
Crystalsorganic versus inorganic
* lattice points need not coincide with atoms
* symmetry can be ‘low’
* unit cell dimensions: 5-50Å, 200-5000Å3
1 Å = 10-10 m = 0.1 nm
Crystals: X-ray diffractiondiffraction: scattering of X-rays by periodic electron densitydiffraction ~ reflection against lattice planes, if: 2dhklsin = n
~ 0.5--2.0ÅCu: 1.54Å
path: 2dhklsin
dhkl
X
Data set:list of intensities I
and angles
Crystalsinformation contained in diffraction data
* How well does the proposed structure correspond to the experimental data? R-factor
consider all (typically 5000) reflections, and comparecalculated structure factors to observed ones.
R = | Fhklobserved - Fhkl
calculated | Fhkl = Ihkl
Fhklobserved
OK if 0.02 < R < 0.06 (small molecules)
* Influence of movement due to temperature: atoms appear ‘smeared out’compared to the static model ADP’s (‘B-factors’).
The R-Factor: Measuring Convergence• To compare the generated electron density map and your
model, you have to use the R-factor.• The R-factor is a measure of convergence between the
intensities given off by your model and the observed intensities.
||Fobs| - |Fcalc||R= ------------------
|Fobs|R:
0.6-VERY BAD
0.5 -BAD
0.4-Recoverable
0.2-Good for Protein
0.05-Good for small organic models
0-PERFECT FIT
Cooling Protein Crystals
• Crystals are damaged by x-ray radiation, resulting in loss of diffraction resolution due to formation of ion radicals or breaking of bonds within the protein. By cooling the crystal to liquid nitrogen temperatures, most of the radiation damage is eliminated. Cryocrystallography is thus an important method in determining protein structures.
• The common method for cooling is flash cooling. The crystal is held in a millimeter diameter loop (see picture) and quickly immersed in liquid nitrogen, liquid propane, or placed under a nitrogen boil-off cold stream.
• Flash cooling helps to prevent ice formation. Stresses during ice formation damage fragile protein crystals. Chemical cryoprotectants added to the solvent further suppress ice formation, keeping the surrounding solution glassy or vitreous.
• Finding the proper cryoprotectant is tedious because it is protein specific. Trial-and-error is the main method, and some crystals don’t have useable cryoprotectants. A general method for cooling without needing crystal specific cryoprotectants would be useful.
Flash cooled crystal embedded in
vitreous solvent.
Pressure and Proteins
• The lab uses pressure to elucidate the structural basis for pressure effects on proteins and to develop a novel method for cooling protein crystals. Pressures used in the lab are a few thousand atmospheres.
• EFFECTS ON PROTEIN STRUCTURE
• Fluctuations and the internal arrangements of atoms plays a crucial in protein function. Both can be probed with pressure. Pressure response of proteins is not compressive and is highly anisotropic. There are internal structural rearrangements.
• EFFECT ON CRYSTAL COOLING
• Pressure is known to slow kinetics of ice formation. Pressure also makes accessible other ice phases which contracts unlike normal hexagonal ice. Thus cooling under pressure might be a general method without needing chemical cryoprotectants.
Alpha helices in myoglobin rearrange when pressurized to 1500 atm. (yellow, 1 atm; green, 1500 atm)
Phase diagram of water.
Various High Pressure Techniques in the Lab
pressure generator
CELL FOR COOLING CRYSTALS UNDER PRESSURE
liquid pressurizing medium
protein crystal
beryllium pressure cell
protein crystal
pressurizingmedium
CELL FOR DIRECT X-RAY CRYSTALLOGRAPHY
UNDER PRESSUREat room temperature
Pressurised gas (blue arrow) is applied from a pump (not
shown).
protein crystal held on a loop using surface tension of solvent
CELL FOR COOLING CRYSTALS UNDER PRESSURE
gas pressurizing medium, cooling direclty onto loop
The Raw Data
Every atom in a unit cell contributes to every reflection
in the diffraction pattern.
Two Pieces of Data• The position of a reflection
point on the reciprocal lattice, given by coordinates h,k,l. Determined by the direction reflected.
• The intensity of the reflection.
From diffraction to electron density map
To get from the diffraction pattern to the electron density, you have to use a Fourier Transform.
Fourier Transform
Once you have an electron density map, you can begin to fit models to it.
Resolution• Resolution: another
measure of how good your model is.
• Resolution gives the size of the smallest molecule you can see or resolve.
• Dependent on the amount of data ultimately phased and used in structure determination.
Limitations and Difficulties, Besides the Phase Problem
• Crystallizing Protein:– Fragile
– Requires a crystal with shortest side 0.2 mm
• Flaws of Crystallization:– Disorder in Unit Cell
– Vibrations of molecules
– Distortion in Crystallization
Fix-Its:
Cryogenic Cooling
Steps of Protein X-ray Crystallography:
Crystallize your protein.
Cryo-freeze your protein.
Do an X-ray diffraction.
Make a heavy atom derivative of protein.
Take X-ray diffraction of the derivative.
Do a Fourier Transform (or let a computer do it).
Create models.
Check R-Factor of models.
Conclusión
Protein x-ray crystallography- practical point of view
A) cloning B) expression
6.5
14.4
21.5
31.0
45.0
66.2 kDa
1
pET3aTBPS1595153bp
TBP
NdeI (4088)
BamHI (4639)
Expression vector: a plasmid carrying the gene of interest
Protein SDS PAGE gel: each band corresponds to one protein
TBP
D) crystallization E) solving the structureC) purification
A protein crystalSDS PAGE showing a purified protein
Ribbon representation of a protein structure (violet) bound to DNA
Protein x-ray crystallography- practical point of view
Some important recent structures and what can we learn from them
K. Luger et al, Nature 1997.
The nucleosome core particle
Aquaporin
(water channel)
H. Sui et al, Nature 2001.
Some important recent structures and what can we learn from them
The ribosome
(large subunit)
N. Ban et al, Science 2000.
Some important recent structures and what can we learn from them
But it sometimes happens …
Rod Casey (Norwich): crystalloid in GM wheat with soya protein
Hydroxyapatite is the primary structural component of bone. As its formula
suggests, it consists of Ca2+ ions
surrounded by PO42– and OH– ions.
Estructura cristalina de la hidroxiapatita
http://www.rcsb.org/pdb/
PDB Protein Data Bank
• Currently contains about 13762 structures of macromolecules – proteins, nucleic acids, protein-DNA complexes and carbohydrates
• 11232 – X-Ray Diffraction & Other
• 2138 – NMR
• 302 – Theoretical Modeling
PDB: Growth
Unidad Asimétrica
