“Structure isn`t everything, but it sure helps” “Light is a

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“Structure isn`t everything, but it sure helps” “Light is a
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“Structure isn’t everything, but it sure helps”
Brian W. Matthews
Biophysical Journal (Annual Meeting Abstracts) 2001
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“Light is a messenger,
carrying a story about the form of the object…”
W. L. Bragg,
Mackenzie Davidson Memorial Lecture
November 14, 1928
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A. de Saint-Exupéry,
1957, «Le Petit Prince», Gallimard,7
Paris
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“ … appreciation of ours present limitation in the area of
protein interactions provides a salutary antidote to the
impression of perfection that the student of biochemistry is
likely to receive from the amount of structural detail of the
proteins that X-ray crystallography, and more recently
magnetic resonance, have made available”
Gregorio Weber
in “Protein Interactions”, 1992
Chapter II - The Chemical Potentials of Proteins
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Cristalomancia
Origem: Wikipédia, a enciclopédia livre.
Cristalomancia é o uso dos cristais ou pedras semipreciosas para supostamente
prever o futuro; podendo ser por meio da uma bola de cristal ou de jogos com
pequenas pedras.A bola de cristal é um instrumento das artes adivinhatórias, muito
popular entre os videntes. A cristalomancia é também muito praticada pelas bruxas,
mais com um propósito filosofico.
Prática dessa mancia
1.Antes de tudo, purifique o cristal que será usado.
2.Relaxe, feche os olhos, tire o peso de seus ombros.
3.Abra os olhos, deixe sua mente ver o que tem dentro do bola.
•
•
•
•
•
Introduction
Crystallization Techniques
Symmetry
Diffraction
Structure elucidation
Lista de fatores
O significado de cada fator dentro da bola de cristal.
•Nuvens Violetas: harmonia e tranqüilidade
•Nuvens Azuis: conquista e felicidade
•Nuvens Verde: lucro e prosperidade
•Nuvens Amarelas: duvidas esclarecidas em breve
•Nuvens
definitivas
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•Nuvens Vermelhas: obstáculos e agitação
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Abordagem moderna / contemporânea de
descoberta e desenvolvimento de fármacos
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RMN
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Intr Macr Xt, McPherson
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Cryo-EM & Single Particle
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2
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Cristalografia e difração de raios-X
2. Medir difração
1. Crescer cristais
3. Resolver fase e
refinar estrutura
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The first crystal structure of a protein molecule
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•© 2006
•Academic
Press
John Kendrew with model of myoglobin in progress. Copyright by
the Laboratory of Molecular Biology in Cambridge, England.
• 1962: Max Ferdinand Perutz and Sir John Cowdery
Kendrew win the Nobel Prize in Chemistry for their
studies on the structures of globlular proteins.
• The structure of myoglobin was solved by MIR.
http://en.wikipedia.org/wiki/John_Kendrew
(Max Perutz, 1914-2002)
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The 2 Å-resolution map of sperm-whale myoglobin was
represented by coloured Meccano-set clips on a forest of vertical
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rods.
(Figure provided by M. F. Perutz)
F10
ITC, IUCr
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http://it.iucr.org/figures/
This is a Kendrew wire model of alcohol dehydrogenase that is
about to undergo a round of rebuilding by Maelle Cambillau.
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http://journals.iucr.org/d/issues/2004/12/01/ba5066/index.html
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•© 2006
•Academic
Press
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•© 2006
•Academic
Press
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Cristalografia
• Breve introdução
• Objetivos
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Cristalização
• Tutorial interativo (Java App) Bragg:
http://www.bmsc.washington.edu/people/merritt/bc530/bragg/
• Cristalização:
– screeninig com fatoriais;
– otimização;
“caixinhas”
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“Hanging drop”
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Cristal
Arranjo Cristalino
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• Estrutra altamente ordenada;
• Alta resolução;
• Precisão da posição dos átomos;
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Padrão de difração
Representação esquemática da fonte de raios X
Detector
Monocromador
Ânodo
Rotátório(Cu)
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Fonte primária
de raios X
Feixe
focalizado
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• raios-X difratados pela densidade
eletrônica
dos átomos da amostra.34
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Mapa de densidade eletrônica
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Mapa de densidade eletrônica
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Resolução
• Qualidade do cristal;
• Certeza da localização
de um grupo químico
no espaço;
(d)
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Cristalização - Métodos
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Soluções para cristalização
kits comerciais
Four major steps in crystallization
• Obtain large amounts of pure protein
samples
• Choose a protein buffer in which the
protein is both soluble and stable
• Bring protein solution to supersaturation
where spontaneous nucleation can take
place
• Crystal growth now begins
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•
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•
Hampton
Jena
Emerald
Qiagem
Sigma
…
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Tutoriais de cristalização
• Rigaku http://www.rigaku.com/protein/crystallization.html
• Hampton http://hamptonresearch.com/experiments.aspx
• Google it !
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Robos de cristalização
• Métodos: Sitting / Capilar / Hanging / Batch
• Robo de preparo de solução
• Métodos de pipetagem: spray / toque
(dispensing)
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Robos de cristalização
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Robos de cristalização
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Robos de cristalização
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Solubility
As a rule, protein solubility will usually increase as you add salt
to your aqueous solution, then begin to decrease when the salt
concentration gets high enough to compete with the protein for
hydration (interaction with water molecules).
HbCO
(carboxy hemoglobin)
solubility as a function
of ionic strength in the
presence of several
different ty pes of salts
Diagram from the website of Alan Clark, Victoria University of Wellington, New Zealand
http://www2.vuw.ac.nz/staff/alan_clark/teaching/index.htm
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Supersaturation
Diagrama de Fase
Supersaturation can be achieved by adding more of a substance
(to a solution) than can normally be dissolved. This is a
thermodynamically unstable state, achieved most often in
protein crystallography by vapor diffusion or other slow
evaporation techniques.
Zone 1 - Metastable zone.
The solution may not nucleate for a long time
but this zone will sustain growth.
It is frequently necessary to add a seed crystal.
Zone 2 - Nucleation zone.
Protein crystals nucleate and grow.
Zone 3 - Precipitation zone.
Proteins do not nucleate but precipitate out
of solution.
Diagram from the website for The University of Reading, Course FS460
Investigating Protein Structure and Function
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Diagrama de Fase
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•© 2006
•Academic
Press
Nucleation
A phenomenon whereby a “ nucleus”, such as a dust particle, a
tiny seed crystal, or commonly in protein crystallography, a
small protein aggregate, starts a crystallization process.
Nucleation poses a large energy barrier, which is easier to
overcome at a higher level of supersaturation.
Common difficulties:
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1. If supersaturation is too high, too many nuclei form, hence
an overabundance of tiny crystals.
2. In supersaturated solutions that don’t experience
spontaneous nucleation, crystal growth often only occurs in
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“ seeds”.
Crystal Growth
Adding single molecules to the
surfaces of the nucleating lattice.
Illustrated here through the work of
Li and Nadarajah of
The Macromolecular
Crystallization Laboratory
at the University of Toledo.
AFM image of individual
lysozyme molecules on
the (110) face of a
tetragonal crystal.
(Li and Nadarajah)
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The growth steps and growth units of
Lysozyme. The growth steps are at least
bimolecular in height. The minimum
growth unit for this step must be a
tetramer corresponding to a single turn of
H. Li, M.A. Perozzo, J.H. Konnert, the 43 helix as shown here.(Nadarajah)
A, Nadarajah & M.L. Pusey, Acta
Crystallographica, D55,
1023-1035 (1999).
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Cessation of growth
Caused by the development of growth defects or the
approach of the solution to equilibrium.
Mother liquor
The solution in which the crystal exists - this is often
not the same as the original crystallization screening
solution, but is instead the solution that exists after
some degree of vapor diffusion, equilibration through
dialysis, or evaporation.
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Major factors that affect crystallization
1) Purity of proteins
2) Protein concentration
3) Starting conditions (make-up of the protein solution)
4) Precipitating agent (precipitant)
5) Temperature
6) pH
7) Additives: Detergents, reducing agents, substrates, co-factors,
etc.
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Intr Macr Xt, McPherson
1) Purity of proteins
Sources of heterogeneity (other than unrelated
proteins and nucleic acids as contaminants):
•
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•
•
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ITC,
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Partial proteolysis products
Oxidation of cysteines
Deamidation of Asn and Gln to Asp and Glu
Post-translational modifications
Oligomerization
Isoforms
Misfolded population
Structural flexibility
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2) Protein concentration
Consistency and reproducibility are the major issues
with protein concentration - you should have a reliable
assay for determining the concentration.
• Extinction coefficient for tryptophan
• Bradford Assay (BSA is used as a standard)
3) Starting conditions (make-up of
the protein solution)
E. coli expression systems are crystallographers’ most
commonly used method of obtaining protein. Problems can
arise from low expression yields:
• Cytotoxic - your protein is killing your E. coli
• Unstable plasmid or mRNA
• Protein is misfolded (coexpress with GroEL?)
• Some common eukaryotic codons are rare in E. coli
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The main point is to KNOW what your starting conditions
are for purposes of reproducibility.
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4) Precipitating agent (precipitant)
Salts
Ammonium sulfate
Sodium chloride
Potassium phosphate
Organic reagents
MPD
Isopropanol
Polyethylene glycol
PEG 4000
PEG 6000
PEG 8000
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Intr Macr Xt, McPherson
5) Temperature
Temperature affects protein stability and also the dynamics of how
protein solution reaching supersaturated states.
Ideally:
•
An individual cry stal screen should be kept at constant temperature
• Each set of conditions should be screened at several temperatures
• The easiest are 4 C and room temperature, also try 12 or 15 C
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6) pH
Surface charges affect “ crystal packing”.
(Crystal packing refers to the spatial arrangement of
molecules within the crystal, particularly in
reference to their relationships to one another.)
Hydrophobic interactions are less important than
electrostatic interactions in crystal packing.
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7) Additives:
Sometimes you can increase the stability of your protein,
and/or the homogeneity of its conformation by having
relevant additives present in the crystal screen:
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Detergents
Reducing agents
Substrates
Co-factors
etc.
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Common Methods for Crystallization:
Still no crystals after thorough
screening. Now what?
Vapor Diffusion
Slow Evaporation
Dialysis
• New constructs
Deletion mutants
Complexes with substrates
Protein complex with Fab fragments
Homologous proteins
Fab
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ITC,
F
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Comparative Studies of Protein
Crystallization by Vapour-Diffusion and
Microbatch Techniques
Acta Crystallographica Section D
Volume 54 Issue 1, Pages 8 – 15
Naomi E.Chayen
http://www3.interscience.wiley.com/cgi
-bin/fulltext/119126302/PDFSTART
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Variáveis
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pH
Temperatura
Tampào / precipitantes / ligantes
Pressão
Método de cristalização
Construção de proteína
Variáveis combinadas
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Intr Macr Xt, McPherson
VD: hanging
• Tradicional
• Dos mais usados
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•© 2006
•Academic
Press
Hanging Drop Vapor Diffusion
Most popular method among
protein cry stallographers.
1. Cry stal screen buffer is the
well solution (0.5 - 1 mL)
2. Drop (on siliconized glass
cover slip) is 1/2 protein
solution, 1/2 cry stal screen
buffer (6-10 L). So, the
concentration of precipitant in
the drop is 1/2 the
concentration in the well.
3. Cover slip is inverted over
the top of the well and sealed
with vacuum grease (airtight).
4. The precipitant concentration in the drop will equilibrate with the
precipitant concentration in the well via vapor diffusion.
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VD: hanging
VD: hanging
CrystalSupport X-Seal
A Standard crystallization support
B The surface of the crystallization support will easily
accommodate 4 drops.
C The screw-in crystallization supports allow easy setup and
reopening.
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http://www.qiagen.com/
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http://www.qiagen.com/
VD: hanging
Support for Multidrop Experiments
Flattened Drops for Easier Visualization
Diagrama esquemático demonstrando o
aparato utilizado para cristalização de
proteínas pelo método da gota pendente.
(arte: Ronaldo Nagem)
A Hanging drop in standard crystallization support side view and top view
B: Hanging drop in Dropguard crystallization
support
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http://www.qiagen.com/
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•© 2006
•Academic
Press
VD: sitting
• Placas
• Interface
• Coleta cristal
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Sitting Drop Vapor Diffusion
Same basic principles
as in hanging drop
method, except the drop
containing y our sample
sits on a bridge within
the well. This allows for
a larger sample size (20 40 L), however protein
is frequently precious to
the cry stallographer, so
there isn’t that much
demand for a larger sample
size.
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Intr Macr Xt, McPherson
VD: sitting
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Intr Macr Xt, McPherson
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VD: sitting
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www.douglas.co.uk
VD: sitting
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www.douglas.co.uk
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www.douglas.co.uk
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VD: sitting
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VD: sitting
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www.douglas.co.uk
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Microbatch
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Crescendo cristais….
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Videos
• - Start with very pure protein
• - Create a supersaturated the solution
• - Wait… mins , days, weeks, …
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reservoir volume ~ 1 mL droplet volume ~ 2L
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Interpreting the Results of the Crystallization Experiment
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Cristal !
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Cristal !
• É proteína ou sal ?
• Não é sal !.....
• Difrata !.........
Proteína !
Difrata ?
É mosaico / twinning ?
• Boa mosaicidade !
• Coletado !
Resiste à coleta ?
Como resolver fase ?
– Subst molec: que modelo usar ?
– Mét. direto:
Se-Met ?
Metais ?
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Difrata ?....
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Resolução
Mosaicidade
- “multiplos cristais”
- quanto menor, melhor (< 1o )
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Difração
- Organização do cristal
- Tamanho (massa)
- Feixe de luz (fluxo de fontos)
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© 2006
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Academic Press
• Qualidade do
cristal;
• Certeza da
localização de um
grupo químico no
espaço;
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Resolução em experimento de difração de cristais de
macromoléculas e aplicabilidade
•
Novos enovelametos
•
Interação com macromoléc ulas:
•
Efeitos conformaci onais
•
Interação com pequenas moléculas (fármacos, etc):
•
Duplas ocupânci as, dinâmica, detalhes de interação intramolec ul ar:
/ estruturas:
< 3.5 Å
< 3.5 Å
de estruturas conhecidas:
< 3.0 Å
< 2.5 Å
<
2.0 Å
•
Refinamento de parâmetros estruturais e validação de metodol ogia:
<
1.5 Å
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http://www.hamptonresearch.com/stuff/gallery.html
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Properties of protein crystals
• Soft, easy to crush
• Contain large solvent channels
– Relatively large organic and inorganic molecules
can diffuse inside
• Anisotropic physical properties
– Birefrigence due to anisotropic refraction indices
• Ability to diffract X-ray due to regular spaced
lattices
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Padrão de difração
Varian (Oxford Diffraction)
Quem é proteína, quem é sal ?!
(corante: azul de metileno,
Izit ™ p/ Hampton)
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• raios-X difratados pela
densidade
eletrônica dos 130
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átomos da amostra.
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http://www.oxford-diffraction.com/pdf/PXScanner_handout.pdf
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The oscillation equipment
Varian (Oxford Diffraction)
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http://www.oxford-diffraction.com/pdf/PXScanner_handout.pdf
Rotates the crystal about an axis () perpendicular to the
x-ray beam (and normal to the goniometer). The diffraction
pattern from a crystal is a 3-D pattern, and the crystal must
be rotated in order to observe all the diffraction spots.
133
This nice diagram also comes from Bernhard Rupp’s Crystallography
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101 website: http://www-structure.llnl.go v/Xr ay/101index. ht ml
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Coleta – montagem de cristal
Crystal Mounting
•
•
•
•
Capillary tubes
(Glass or Quartz)
Capilares
Loop
Pás
Coleta direto do aparato
Cryo-loops
(thin nylon)
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Capilar
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•© 2006
•Academic
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Princ Ptn x-ray diffr,
Drenth, 3r d Ed139
141
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http://it.iucr.org/figures/Fafig5o1o2o2.gif
140
142
Intr Macr Xt, McPherson
23
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146
Preparo de cristais para coleta
•
•
•
•
•
Soaking com tampão-mãe – retirar tp ptn
Soaking com ligante
Soaking com crioprotetor / óleo
Quebra
Remoção de ‘pele’ / microcristais
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F10
ITC, 148
IUCr
24
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Coleta a RT:
MiTeGen MicroRT™ Room
Temperature Mounting System
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Criocristalografia
• Desenvolvido em
• Capilar
• Loop em capilar
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Água / gelo
Hkl indeces, Bragg spacings d and relative intensities I/Io of reflections observed in powder
diffraction from crystals of hexagonal ice at 98 K as reported by Dowell and Rinfret
(1960)
Note that the relative intensities of the ice rings found in diffraction photographs form
macromolecular crystals often deviate substantially from the values given in the table
hkl
100
002
101
102
110
103
200
112
201
202
D (Å)
3.897
3.667
3.441
2.671
2.249
2.072
1.948
1.918
1.883
1.721
I/Io
100
75
53
17
39
30
4
18
3
2
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ITC, IUCr
160
http://it.iucr.org/figures/
26
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Recommended pathways for optimizing cryoprotectant
conditions and flash cooling
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ITC, IUCr
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http://it.iucr.org/figures/
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ITC, IUCr
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http://it.iucr.org/figures/
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ITC, IUCr
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http://it.iucr.org/figures/
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ITC, IUCr
164
http://it.iucr.org/figures/
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ITC, IUCr
165
http://it.iucr.org/figures/
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Sistemas criogênicos
Soprador de N2g
• a partir de N2L
• compressão de N2g do ar
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Sistemas criogênicos
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Sistemas criogênicos
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Crio vs RT
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Otimização de cristalização
• Vantagem: não usa aditivo crioprotetor,
não ‘congela’ cristal, dispensa acessório
criogênico
• Desvantagem: baixa difração, dano por
radiólise
• Refinamento das condições: elementos precipitantes
(tãmponante, pH, aditivos), temperatura, método
• Otimizar a pureza do material: preparação proteica,
reagentes
• Trocar o suporte (plaquinha cristalográfica - efeito de
superfície)
• Seeding
• Ligantes: estabilizantes da proteína e/ou interação
interproteica (rede cristalográfica)
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Coleta – fontes de raios-X
•
•
•
•
Anodo
Tubo selado
Sincrotron
Fita adesiva
(http://www.youtube.com/watch?v=LQBjR
F9mX1Y)
• Coleta remota / in situ
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LNLS
INMETRO
Cu
Mo
Diffraction directly from
cry stallization screening plates
Single cry stal diffraction
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Dual wavelength
187webs ite
Images from Agilent
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anodo rotatório
… o rder a
d i f fracto meter
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31
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Geradores: Funciomanento
• Tubo Selado
• Anodo rotatório
• Sincrotron
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199
Intr Macr Xt, McPherson
ITC,201F
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Drenth, 3r d Ed200
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Drenth, 3r d Ed202
•© 2006
•Academic
Press
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Intr Macr Xt, McPherson
Rigaku
•
•
•
•
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•© 2006
•Academic
Press
Anodo rotatório
Tubo selado
Ambos, opções Cu, Mo
Detector RaxisIV (Rigaku) ou Mar – IP /
CCD
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Bruker - AXS
• Tubo selado
• Detectores: CCD
• Fontes: Cu / M o
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Intr Macr Xt, McPherson
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34
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Agilent (Oxford Diffraction)
Bruker - AXS
• Tubo selado
• Detectores: CCD
• Fontes: Cu / Mo
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Agilent (Oxford Diffraction)
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Agilent (Oxford Diffraction)
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The LNLS 1.37 GeV electron storage in December 7, 1996 [3]. All twelve
dipolar magnets are visible. Inside the ring two klystrons and their
associated modulators are apparent; they feed the LINAC located
underground with RF
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Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997
35
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Brazilian Sy nchrotron Light Laboratory (LNLS)
Campinas - Brazil
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ITC,
F
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17 Fev 2004
(ainda um pouco oscilante…)
Typical time dependence of electron current in the LNLS storage ring
(April 1997).
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Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997
Photon flux from the bending magnets of the storage ring and from the planned
7 Tesla wavelength-shifter.
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Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997
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http://www.scielo.br/scielo.php?pid=S1516-14392002000100002&script=sci_arttext
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Mat. Res. vol.5 no.1 São Carlos Jan./Mar. 2002
36
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Picture of the SAXS beamline(3). The beamline passes across the shielding at the left. The sequence
of optical components is: mirror chamber (for vertical focusing), first four-slit set, monochromator
chamber (for monochromatization and horizontal focusing), second four-slit set, guard slit set, sample
holder, beamstopper and vertical X-ray position-sensitive detector.
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Location of the nine beamlines to be opened to users in 1997 (seven) and 1998 (two).
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222
Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997
LNLS: general considerations
Open facility supported by the Brazilian Science
and Technology Ministery (MCT).
Beginning of operation: July 1997.
Link: http:// www.lnls.br
Linac: 120 MeV (e - injection energy )
Booster: 500 MeV (maximum e - operation energy )
Storage Ring : 1.37 GeV (e - operation energy )
D02A-SAXS2
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L. A. Bernardes
12 Bending magnet (BM) beamlines open for users:
9 in X-ray range, 3 in UV and soft X-ray range.
3 BM beamlines under commissioning.
1 Wiggler beamline under commissioning for
anomalous diffraction protein crystallography.
2 New insertion device beamlines under construction:
ondulator beamline for UV experiments and wiggler
beamline for material science (X-ray experiments).
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SAXS – SAS1
•
•
•
•
•
Primeira linha do LNLS
Agora em outra saída do anel
Novo detector – 2D
Mesmo desenho da MX1
Detalhes da linha antiga:
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37
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SAXS – SAS1
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Mat. Res. vol.5 no.1 São Carlos Jan./Mar. 2002
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L. A. Bernardes ©
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230
L. A. Bernardes ©
Detectores: Funcionamento
• Placa de imagem
• CCD
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L. A. Bernardes ©
38
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Rigaku Ultra18X (IFSC, LNLS)
Image Plate Mar345dtb
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IP - M ar345dtb
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M X1 – M arCCD
M ardtb
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IP – R-AXIS V
IP - M ar345dtb
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M X1 – M arCCD
The SX Series are the only
CCD X-ray detectors that are ideal for
both sy nchrotrons and rotating anode Xray sources. The SX-165 features a
round, 165 mm diameter active area,
and a versatile, high-resolution CCD
chip.
The CCD chip in the SX-165
is cooled to -70°C and protected inside
a sealed vacuum chamber. The resulting
dark current, less than 0.01e-/pixel/sec.,
allows exposures long enough for data
collection from any crystal on any Xray source. The refrigeration system
requires only a standard electrical outlet
and no cooling water.
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•© 2006
•Academic
Press
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http://www.mar-usa.com/support/downloads/sx_series.pdf
M X2 - M arMosaic
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http://www.marresearch.com/marccd.ht m
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M X2 - M arMosaic
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V POSLATAM -http://www.marresearch.com/products.mx-series.html
Buzios
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Quantum
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40
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•Academic
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M esa detectora
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