PowerPoint Presentation · 2018-07-22 · pequenas pedras.A bola de cristal é um instrumento das...
Transcript of PowerPoint Presentation · 2018-07-22 · pequenas pedras.A bola de cristal é um instrumento das...
10/22/2012
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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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A. de Saint-Exupéry, 1957, «Le Petit Prince», Gallimard,
Paris
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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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“ … 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.
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 Laranjas: decisões difíceis definitivas •Nuvens Vermelhas: obstáculos e agitação
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• Introduction • Crystallization Techniques
• Symmetry • Diffraction
• Structure elucidation
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Abordagem moderna / contemporânea de
descoberta e desenvolvimento de fármacos
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RMN Cryo-EM & Single Particle
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1. Crescer cristais
3. Resolver fase e refinar estrutura
2. Medir difração
Cristalografia e difração de raios-X
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•Academic
Press
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The first crystal structure of a protein molecule
• 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.
(Max Perutz, 1914-2002)
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http://en.wikipedia.org/wiki/John_Kendrew
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ITC, IUCr
http://it.iucr.org/figures/
John Kendrew with model of myoglobin in progress. Copyright by
the Laboratory of Molecular Biology in Cambridge, England.
The 2 Å-resolution map of sperm-whale myoglobin was
represented by coloured Meccano-set clips on a forest of vertical rods. (Figure provided by M. F. Perutz) 10/22/2012 20 V POSLATAM - Buzios
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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•© 2006
•Academic
Press
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•© 2006
•Academic
Press
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Cristalografia
• Breve introdução
• Objetivos
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• Tutorial interativo (Java App) Bragg: http://www.bmsc.washington.edu/people/merritt/bc530/bragg/
• Tutorial interativo (Java App) Bragg: http://www.bmsc.washington.edu/people/merritt/bc530/bragg/
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Cristalização
• Cristalização:
– screeninig com fatoriais;
– otimização;
“Hanging drop” “caixinhas”
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Arranjo Cristalino
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Cristal
• Estrutra altamente ordenada;
• Alta resolução;
• Precisão da posição dos átomos;
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Representação esquemática da fonte de raios X
Ânodo
Rotátório(Cu)
Fonte primária
de raios X
Monocromador
Feixe
focalizado
Detector
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Padrão de difração
• raios-X difratados pela densidade eletrônica dos átomos da amostra. 10/22/2012 34 V POSLATAM - Buzios
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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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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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Soluções para cristalização
kits comerciais
• Hampton
• Jena
• Emerald
• Qiagem
• Sigma
• …
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Tutoriais de cristalização
• Rigaku -
http://www.rigaku.com/protein/crystall ization.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).
Diagram from the website of Alan Clark, Victoria University of Wellington, New Zealand
http://www2.vuw.ac.nz/staff/alan_clark/teaching/index.htm
HbCO
(carboxy hemoglobin)
solubility as a function
of ionic strength in the
presence of several
different ty pes of salts
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Supersaturation
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
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Diagrama de Fase
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Diagrama de Fase
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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:
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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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)
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
the 43 helix as shown here.(Nadarajah) H. Li, M.A. Perozzo, J.H. Konnert, A, Nadarajah & M.L. Pusey, Acta Crystallographica, D55,
1023-1035 (1999). 10/22/2012 69 V POSLATAM - Buzios
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
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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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1) Purity of proteins
Sources of heterogeneity (other than unrelated
proteins and nucleic acids as contaminants):
• 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)
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 10/22/2012 75 V POSLATAM - Buzios
3) Starting conditions (make-up of
the protein solution)
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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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:
• Detergents
• Reducing agents
• Substrates
• Co-factors
• etc.
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Still no crystals after thorough
screening. Now what?
• New constructs
Deletion mutants
Complexes with substrates
Protein complex with Fab fragments
Homologous proteins
Fab
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Common Methods for Crystallization:
Vapor Diffusion
Slow Evaporation
Dialysis
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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
• pH
• Temperatura
• Tampào / precipitantes / ligantes
• Pressão
• Método de cristalização
• Construção de proteína
• Variáveis combinadas
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• Tradicional
• Dos mais usados
VD: hanging
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•Academic
Press
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Hanging Drop Vapor Diffusion
Most popular method among
protein cry stallographers. 1. Crystal 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
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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VD: hanging
http://www.qiagen.com/
CrystalSupport X-Seal
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VD: hanging
A Hanging drop in standard crystallization support -
side view and top view
B: Hanging drop in Dropguard crystallization
support
Flattened Drops for Easier Visualization Support for Multidrop Experiments
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Diagrama esquemático demonstrando o
aparato utilizado para cristalização de
proteínas pelo método da gota pendente.
(arte: Ronaldo Nagem)
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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 your 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 crystallographer, so
there isn’t that much
demand for a larger sample
size.
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VD: sitting
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VD: sitting
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VD: sitting
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VD: sitting
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VD: sitting
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Microbatch
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• - Start with very pure protein
• - Create a supersaturated the solution
• - Wait… mins , days, weeks, …
reservoir volume ~ 1 mL droplet volume ~ 2L
Crescendo cristais….
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Videos
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Interpreting the Results of the Crystallization Experiment
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Cristal !
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• É proteína ou sal ? Proteína !
• Não é sal !..... Difrata ?
• Difrata !......... É mosaico / twinning ?
• Boa mosaicidade ! Resiste à coleta ?
• Coletado ! Como resolver fase ?
– Subst molec: que modelo usar ?
– Mét. direto: Se-Met ? Metais ? Difrata ?....
Cristal !
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© 2006
Academic Press
Difração
- Organização do cristal
- Tamanho (massa)
- Feixe de luz (fluxo de fontos)
Mosaicidade
- “multiplos cristais”
- quanto menor, melhor (< 1o)
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Resolução
• Qualidade do cristal;
• Certeza da localização de um grupo químico no espaço;
(d)
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Resolução em experimento de difração de cristais de
macromoléculas e aplicabilidade
• Novos enovelametos / estruturas: < 3.5 Å
• Interação com macromoléculas: < 3.5 Å
• Efeitos conformaci onais de estruturas conhecidas: < 3.0 Å
• Interação com pequenas moléculas (fármacos, etc): < 2.5 Å
• Duplas ocupânci as, dinâmica, detalhes de interação intramolecul ar: <
2.0 Å
• Refinamento de parâmetros estruturais e validação de metodol ogia: <
1.5 Å
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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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Varian (Oxford Diffraction)
Quem é proteína, quem é sal ?! (corante: azul de metileno, Izit ™ p/ Hampton)
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Padrão de difração
• raios-X difratados pela densidade eletrônica dos átomos da amostra.
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http://www.oxford-diffraction.com/pdf/PXScanner_handout.pdf 10/22/2012 131 V POSLATAM - Buzios
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Varian (Oxford Diffraction)
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The oscillation equipment 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.
This nice diagram also comes from Bernhard Rupp’s Crystallography
101 website: http://www-structure.llnl.gov/Xr ay/101index.ht ml 10/22/2012 134 V POSLATAM - Buzios
Crystal Mounting
Capillary tubes
(Glass or Quartz)
Cryo-loops
(thin nylon)
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Coleta – montagem de cristal
• Capilares
• Loop
• Pás
• Coleta direto do aparato
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•© 2006
•Academic
Press
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Capilar
http://it.iucr.org/figures/Fafig5o1o2o2.gif 10/22/2012 138 V POSLATAM - Buzios
Princ Ptn x-ray diffr,
Drenth, 3rd Ed 10/22/2012 139 V POSLATAM - Buzios 10/22/2012 140 V POSLATAM - Buzios
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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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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 D (Å) I/Io
100 3.897 100
002 3.667 75
101 3.441 53
102 2.671 17
110 2.249 39
103 2.072 30
200 1.948 4
112 1.918 18
201 1.883 3
202 1.721 2
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F10
ITC, IUCr
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Recommended pathways for optimizing cryoprotectant
conditions and flash cooling
F10
ITC, IUCr
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F10
ITC, IUCr
http://it.iucr.org/figures/ 10/22/2012 162 V POSLATAM - Buzios
F10
ITC, IUCr
http://it.iucr.org/figures/ 10/22/2012 163 V POSLATAM - Buzios
F10
ITC, IUCr
http://it.iucr.org/figures/ 10/22/2012 164 V POSLATAM - Buzios
F10
ITC, IUCr
http://it.iucr.org/figures/ 10/22/2012 165 V POSLATAM - Buzios 10/22/2012 166 V POSLATAM - Buzios
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10/22/2012 167 V POSLATAM - Buzios 10/22/2012 168 V POSLATAM - Buzios
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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
• Vantagem: não usa aditivo crioprotetor,
não ‘congela’ cristal, dispensa acessório criogênico
• Desvantagem: baixa difração, dano por
radiólise
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Otimização de cristalização
• 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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INMETRO
Diffraction directly from
cry stallization screening plates
Images from Agilent webs ite
Cu Mo
Single cry stal diffraction Dual wavelength
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LNLS
anodo rotatório 10/22/2012 188 V POSLATAM - Buzios
… o rder a
di f fractometer
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Geradores: Funciomanento
• Tubo Selado
• Anodo rotatório
• Sincrotron
10/22/2012 197 V POSLATAM - Buzios •© 2006
•Academic
Press
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Intr Macr Xt, McPherson 10/22/2012 199 V POSLATAM - Buzios
Princ Ptn x-ray diffr,
Drenth, 3rd Ed 10/22/2012 200 V POSLATAM - Buzios
ITC, F 10/22/2012 201 V POSLATAM - Buzios Princ Ptn x-ray diffr,
Drenth, 3rd Ed 10/22/2012 202 V POSLATAM - Buzios
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Princ Ptn x-ray diffr,
Drenth, 3rd Ed 10/22/2012 203 V POSLATAM - Buzios Intr Macr Xt, McPherson
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•© 2006
•Academic
Press
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Rigaku
• Anodo rotatório
• Tubo selado
• Ambos, opções Cu, Mo
• Detector RaxisIV (Rigaku) ou Mar – IP / CCD
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Intr Macr Xt, McPherson 10/22/2012 207 V POSLATAM - Buzios
Bruker - AXS
• Tubo selado
• Detectores: CCD
• Fontes: Cu / Mo
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Bruker - AXS
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Agilent (Oxford Diffraction)
• 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
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-97331997000400002
Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997 10/22/2012 214 V POSLATAM - Buzios
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Brazilian Synchrotron Light Laboratory (LNLS)
Campinas - Brazil
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Typical time dependence of electron current in the LNLS storage ring (April 1997).
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-97331997000400002
Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997 10/22/2012 217 V POSLATAM - Buzios
17 Fev 2004 (ainda um pouco oscilante…)
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Photon flux from the bending magnets of the storage ring and from the planned 7 Tesla wavelength-shifter.
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-97331997000400002
Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997 10/22/2012 219 V POSLATAM - Buzios http://www.scielo.br/scielo.php?pid=S1516-14392002000100002&script=sci_arttext
Mat. Res. vol.5 no.1 São Carlos Jan./Mar. 2002 10/22/2012 220 V POSLATAM - Buzios
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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.
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-97331997000400002
Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997 10/22/2012 221 V POSLATAM - Buzios
Location of the nine beamlines to be opened to users in 1997 (seven) and 1998 (two).
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-97331997000400002
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L. A. Bernardes © 10/22/2012 223 V POSLATAM - Buzios
LNLS: general considerations
Linac: 120 MeV (e - injection energy )
Booster: 500 MeV (maximum e - operation energy )
Storage Ring : 1.37 GeV (e - operation energy )
Open facility supported by the Brazilian Science
and Technology Ministery (MCT).
Beginning of operation: July 1997.
Link: http:// www.lnls.br
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). D02A-SAXS2
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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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SAXS – SAS1
http://www.scielo.br/scielo.php?pid=S1516-14392002000100002&script=sci_arttext
Mat. Res. vol.5 no.1 São Carlos Jan./Mar. 2002 10/22/2012 227 V POSLATAM - Buzios
L. A. Bernardes ©
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L. A. Bernardes ©
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L. A. Bernardes ©
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L. A. Bernardes ©
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Detectores: Funcionamento
• Placa de imagem
• CCD
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Rigaku Ultra18X (IFSC, LNLS)
Image Plate Mar345dtb
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IP - Mar345dtb
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IP - Mar345dtb
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IP – R-AXIS V
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Mardtb
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MX1 – MarCCD
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•© 2006
•Academic
Press
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The SX Series are the only
CCD X-ray detectors that are ideal for both synchrotrons and rotating anode X-ray 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 X-ray source. The refrigeration system requires only a standard electrical outlet
and no cooling water.
http://www.mar-usa.com/support/downloads/sx_series.pdf
MX1 – MarCCD
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http://www.marresearch.com/marccd.htm 10/22/2012 241 V POSLATAM - Buzios
MX2 - MarMosaic
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MX2 - MarMosaic
http://www.marresearch.com/products.mx-series.html 10/22/2012 243 V POSLATAM - Buzios
Quantum
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