“Structure isn`t everything, but it sure helps” “Light is a
January 11, 2018 | Author: Anonymous | Category: N/A
Short Description
Download “Structure isn`t everything, but it sure helps” “Light is a...
Description
10/22/2012
“Structure isn’t everything, but it sure helps”
Brian W. Matthews Biophysical Journal (Annual Meeting Abstracts) 2001
10/22/2012
V POSLATAM - Buzios
5
10/22/2012
V POSLATAM - Buzios
6
“Light is a messenger, carrying a story about the form of the object…”
W. L. Bragg, Mackenzie Davidson Memorial Lecture November 14, 1928
10/22/2012
V POSLATAM - Buzios A. de Saint-Exupéry, 1957, «Le Petit Prince», Gallimard,7 Paris
10/22/2012
V POSLATAM - Buzios
8
“ … 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
10/22/2012
V POSLATAM - Buzios
9
10/22/2012
V POSLATAM - Buzios
10
1
10/22/2012
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 10/22/2012Laranjas: decisões difíceis V POSLATAM - Buzios •Nuvens Vermelhas: obstáculos e agitação
11
10/22/2012
V POSLATAM - Buzios
12
Abordagem moderna / contemporânea de descoberta e desenvolvimento de fármacos
10/22/2012
RMN
10/22/2012
V POSLATAM - Buzios
13
Intr Macr Xt, McPherson
10/22/2012
V POSLATAM - Buzios
14
10/22/2012
V POSLATAM - Buzios
16
Cryo-EM & Single Particle
V POSLATAM - Buzios
15
2
10/22/2012
Cristalografia e difração de raios-X
2. Medir difração
1. Crescer cristais
3. Resolver fase e refinar estrutura
10/22/2012
V POSLATAM - Buzios
17
The first crystal structure of a protein molecule
10/22/2012
V POSLATAM - Buzios
18
•© 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)
10/22/2012
V POSLATAM - Buzios
19
The 2 Å-resolution map of sperm-whale myoglobin was represented by coloured Meccano-set clips on a forest of vertical 10/22/2012 V POSLATAM - Buzios rods. (Figure provided by M. F. Perutz)
F10 ITC, IUCr 20 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.
10/22/2012
V POSLATAM - Buzios
21
http://journals.iucr.org/d/issues/2004/12/01/ba5066/index.html
10/22/2012
V POSLATAM - Buzios
22
•© 2006 •Academic Press
3
10/22/2012
10/22/2012
V POSLATAM - Buzios
•© 2006 •Academic Press
23
10/22/2012
V POSLATAM - Buzios
24
10/22/2012
V POSLATAM - Buzios
25
10/22/2012
V POSLATAM - Buzios
26
Cristalografia • Breve introdução • Objetivos
10/22/2012
V POSLATAM - Buzios
27
10/22/2012
V POSLATAM - Buzios
28
4
10/22/2012
Cristalização
• Tutorial interativo (Java App) Bragg: http://www.bmsc.washington.edu/people/merritt/bc530/bragg/
• Cristalização: – screeninig com fatoriais;
– otimização;
“caixinhas”
10/22/2012
V POSLATAM - Buzios
29
10/22/2012
“Hanging drop”
V POSLATAM - Buzios
30
Cristal
Arranjo Cristalino
10/22/2012
• Estrutra altamente ordenada; • Alta resolução; • Precisão da posição dos átomos;
V POSLATAM - Buzios
31
10/22/2012
V POSLATAM - Buzios
32
Padrão de difração Representação esquemática da fonte de raios X Detector
Monocromador
Ânodo Rotátório(Cu)
10/22/2012
Fonte primária de raios X
Feixe focalizado
V POSLATAM - Buzios
33
10/22/2012
• raios-X difratados pela densidade eletrônica dos átomos da amostra.34 V POSLATAM - Buzios
5
10/22/2012
Mapa de densidade eletrônica
10/22/2012
V POSLATAM - Buzios
Mapa de densidade eletrônica
35
10/22/2012
V POSLATAM - Buzios
36
37
10/22/2012
V POSLATAM - Buzios
38
Resolução • Qualidade do cristal; • Certeza da localização de um grupo químico no espaço; (d)
10/22/2012
V POSLATAM - Buzios
Cristalização - Métodos
10/22/2012
V POSLATAM - Buzios
39
10/22/2012
V POSLATAM - Buzios
40
6
10/22/2012
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
• • • • • •
Hampton Jena Emerald Qiagem Sigma …
10/22/2012
V POSLATAM - Buzios
41
10/22/2012
V POSLATAM - Buzios
42
10/22/2012
V POSLATAM - Buzios
43
10/22/2012
V POSLATAM - Buzios
44
10/22/2012
V POSLATAM - Buzios
45
10/22/2012
V POSLATAM - Buzios
46
7
10/22/2012
10/22/2012
V POSLATAM - Buzios
47
10/22/2012
V POSLATAM - Buzios
48
Tutoriais de cristalização • Rigaku http://www.rigaku.com/protein/crystallization.html
• Hampton http://hamptonresearch.com/experiments.aspx • Google it !
10/22/2012
V POSLATAM - Buzios
49
10/22/2012
V POSLATAM - Buzios
50
10/22/2012
V POSLATAM - Buzios
51
10/22/2012
V POSLATAM - Buzios
52
8
10/22/2012
Robos de cristalização • Métodos: Sitting / Capilar / Hanging / Batch • Robo de preparo de solução • Métodos de pipetagem: spray / toque (dispensing)
10/22/2012
V POSLATAM - Buzios
53
10/22/2012
V POSLATAM - Buzios
54
Robos de cristalização
10/22/2012
V POSLATAM - Buzios
55
10/22/2012
Robos de cristalização
10/22/2012
V POSLATAM - Buzios
V POSLATAM - Buzios
56
Robos de cristalização
57
10/22/2012
V POSLATAM - Buzios
58
9
10/22/2012
10/22/2012
V POSLATAM - Buzios
59
10/22/2012
V POSLATAM - Buzios
10/22/2012
V POSLATAM - Buzios
60
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 10/22/2012
V POSLATAM - Buzios
61
62
Intr Macr Xt, McPherson
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
10/22/2012
V POSLATAM - Buzios
63
10/22/2012
V POSLATAM - Buzios
64
10
10/22/2012
Diagrama de Fase
10/22/2012
V POSLATAM - Buzios
65
10/22/2012
V POSLATAM - Buzios
66
•© 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:
10/22/2012
V POSLATAM - Buzios
67
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 10/22/2012 68 the presence of addedV POSLATAM nuclei or- Buzios “ 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)
10/22/2012
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). V POSLATAM - Buzios 69
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. 10/22/2012
V POSLATAM - Buzios
70
11
10/22/2012
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. 10/22/2012
V POSLATAM - Buzios
71
10/22/2012
V POSLATAM - Buzios
72
Intr Macr Xt, McPherson
1) Purity of proteins Sources of heterogeneity (other than unrelated proteins and nucleic acids as contaminants): • • • • • • • • 10/22/2012
V POSLATAM - Buzios
73 F ITC,
10/22/2012
Partial proteolysis products Oxidation of cysteines Deamidation of Asn and Gln to Asp and Glu Post-translational modifications Oligomerization Isoforms Misfolded population Structural flexibility V POSLATAM - Buzios
74
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 10/22/2012
V POSLATAM - Buzios
The main point is to KNOW what your starting conditions are for purposes of reproducibility.
75
10/22/2012
V POSLATAM - Buzios
76
12
10/22/2012
4) Precipitating agent (precipitant) Salts Ammonium sulfate Sodium chloride Potassium phosphate Organic reagents MPD Isopropanol Polyethylene glycol PEG 4000 PEG 6000 PEG 8000 10/22/2012
V POSLATAM - Buzios
77
10/22/2012
V POSLATAM - Buzios
78
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
10/22/2012
V POSLATAM - Buzios
79
10/22/2012
V POSLATAM - Buzios
81
10/22/2012
V POSLATAM - Buzios
Intr Macr Xt, McPherson
80
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.
10/22/2012
V POSLATAM - Buzios
82
Intr Macr Xt, McPherson
13
10/22/2012
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:
• • • • • 10/22/2012
Detergents Reducing agents Substrates Co-factors etc. V POSLATAM - Buzios
83
10/22/2012
V POSLATAM - Buzios
84
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 10/22/2012
10/22/2012
V POSLATAM - Buzios
V POSLATAM - Buzios
85
87
Intr Macr Xt, McPherson
10/22/2012 ITC, F
10/22/2012
V POSLATAM - Buzios
V POSLATAM - Buzios
86
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 88
14
10/22/2012
Variáveis • • • • • • •
pH Temperatura Tampào / precipitantes / ligantes Pressão Método de cristalização Construção de proteína Variáveis combinadas
10/22/2012
V POSLATAM - Buzios
89
10/22/2012
V POSLATAM - Buzios
91
10/22/2012
V POSLATAM - Buzios
10/22/2012
V POSLATAM - Buzios
90
Intr Macr Xt, McPherson
VD: hanging • Tradicional • Dos mais usados
10/22/2012
V POSLATAM - Buzios
92
•© 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. 10/22/2012
V POSLATAM - Buzios
93
94
Intr Macr Xt, McPherson
15
10/22/2012
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.
10/22/2012
V POSLATAM - Buzios
95 http://www.qiagen.com/
10/22/2012
V POSLATAM - Buzios
96 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 10/22/2012
V POSLATAM - Buzios
97 http://www.qiagen.com/
10/22/2012
V POSLATAM - Buzios
98
10/22/2012
V POSLATAM - Buzios
•© 2006 •Academic Press
VD: sitting • Placas • Interface • Coleta cristal
10/22/2012
V POSLATAM - Buzios
99
100
16
10/22/2012
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. 10/22/2012
V POSLATAM - Buzios
101
10/22/2012
V POSLATAM - Buzios
102
Intr Macr Xt, McPherson
VD: sitting
10/22/2012
V POSLATAM - Buzios
103
Intr Macr Xt, McPherson
10/22/2012
VD: sitting
10/22/2012
V POSLATAM - Buzios
V POSLATAM - Buzios
104
www.douglas.co.uk
VD: sitting
105
www.douglas.co.uk
10/22/2012
V POSLATAM - Buzios
106
www.douglas.co.uk
17
10/22/2012
VD: sitting
10/22/2012
V POSLATAM - Buzios
10/22/2012
10/22/2012
VD: sitting
107
10/22/2012
V POSLATAM - Buzios
V POSLATAM - Buzios
109
10/22/2012
V POSLATAM - Buzios
110
V POSLATAM - Buzios
111
10/22/2012
V POSLATAM - Buzios
112
www.douglas.co.uk
108
www.douglas.co.uk
18
10/22/2012
10/22/2012
V POSLATAM - Buzios
113
10/22/2012
V POSLATAM - Buzios
114
Microbatch
10/22/2012
V POSLATAM - Buzios
115
10/22/2012
Crescendo cristais….
V POSLATAM - Buzios
116
Videos
• - Start with very pure protein • - Create a supersaturated the solution • - Wait… mins , days, weeks, …
10/22/2012 V POSLATAM - Buzios reservoir volume ~ 1 mL droplet volume ~ 2L
117
10/22/2012
V POSLATAM - Buzios
118
19
10/22/2012
Interpreting the Results of the Crystallization Experiment
10/22/2012
V POSLATAM - Buzios
119
Cristal !
10/22/2012
V POSLATAM - Buzios
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 ?
10/22/2012
120
V POSLATAM - Buzios
121
10/22/2012
V POSLATAM - Buzios
Difrata ?....
122
Resolução
Mosaicidade - “multiplos cristais” - quanto menor, melhor (< 1o )
10/22/2012
Difração - Organização do cristal - Tamanho (massa) - Feixe de luz (fluxo de fontos)
V POSLATAM - Buzios
© 2006 123 Academic Press
• Qualidade do cristal; • Certeza da localização de um grupo químico no espaço;
10/22/2012
(d)
V POSLATAM - Buzios
124
20
10/22/2012
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
10/22/2012
V POSLATAM - Buzios
125
10/22/2012
http://www.hamptonresearch.com/stuff/gallery.html V POSLATAM - Buzios 126
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 10/22/2012
V POSLATAM - Buzios
127
10/22/2012
128
Padrão de difração
Varian (Oxford Diffraction)
Quem é proteína, quem é sal ?! (corante: azul de metileno, Izit ™ p/ Hampton) 10/22/2012 V POSLATAM - Buzios
V POSLATAM - Buzios
129
10/22/2012
• raios-X difratados pela densidade eletrônica dos 130 V POSLATAM - Buzios átomos da amostra.
21
10/22/2012
10/22/2012
V POSLATAM - Buzios 131 http://www.oxford-diffraction.com/pdf/PXScanner_handout.pdf
10/22/2012
The oscillation equipment
Varian (Oxford Diffraction)
10/22/2012
V POSLATAM - Buzios
V POSLATAM - Buzios 132 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 V POSLATAM - Buzios 134 101 website: http://www-structure.llnl.go v/Xr ay/101index. ht ml
10/22/2012
Coleta – montagem de cristal
Crystal Mounting • • • •
Capillary tubes (Glass or Quartz)
Capilares Loop Pás Coleta direto do aparato
Cryo-loops (thin nylon) 10/22/2012
V POSLATAM - Buzios
135
10/22/2012
V POSLATAM - Buzios
136
22
10/22/2012
Capilar
10/22/2012
V POSLATAM - Buzios
10/22/2012
V POSLATAM - Buzios
10/22/2012
V POSLATAM - Buzios
137
•© 2006 •Academic Press
Princ Ptn x-ray diffr, Drenth, 3r d Ed139
141
10/22/2012
V POSLATAM - Buzios
10/22/2012
V POSLATAM - Buzios
10/22/2012
V POSLATAM - Buzios
138
http://it.iucr.org/figures/Fafig5o1o2o2.gif
140
142 Intr Macr Xt, McPherson
23
10/22/2012
10/22/2012
V POSLATAM - Buzios
143
10/22/2012
V POSLATAM - Buzios
144
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
10/22/2012
V POSLATAM - Buzios
145
10/22/2012
V POSLATAM - Buzios
10/22/2012
V POSLATAM - Buzios
147
10/22/2012
V POSLATAM - Buzios
F10 ITC, 148 IUCr
24
10/22/2012
Coleta a RT: MiTeGen MicroRT™ Room Temperature Mounting System
10/22/2012
V POSLATAM - Buzios
149
10/22/2012
V POSLATAM - Buzios
150
Criocristalografia • Desenvolvido em • Capilar • Loop em capilar
10/22/2012
V POSLATAM - Buzios
151
10/22/2012
V POSLATAM - Buzios
152
10/22/2012
V POSLATAM - Buzios
153
10/22/2012
V POSLATAM - Buzios
154
25
10/22/2012
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
10/22/2012
V POSLATAM - Buzios
155
10/22/2012
V POSLATAM - Buzios
156
10/22/2012
V POSLATAM - Buzios
157
10/22/2012
V POSLATAM - Buzios
158
10/22/2012
V POSLATAM - Buzios
159
10/22/2012
V POSLATAM - Buzios
F10 ITC, IUCr 160 http://it.iucr.org/figures/
26
10/22/2012
Recommended pathways for optimizing cryoprotectant conditions and flash cooling
10/22/2012
V POSLATAM - Buzios
F10 ITC, IUCr 161 http://it.iucr.org/figures/
10/22/2012
V POSLATAM - Buzios
F10 ITC, IUCr 162 http://it.iucr.org/figures/
10/22/2012
V POSLATAM - Buzios
F10 ITC, IUCr 163 http://it.iucr.org/figures/
10/22/2012
V POSLATAM - Buzios
F10 ITC, IUCr 164 http://it.iucr.org/figures/
10/22/2012
V POSLATAM - Buzios
F10 ITC, IUCr 165 http://it.iucr.org/figures/
10/22/2012
V POSLATAM - Buzios
166
27
10/22/2012
10/22/2012
V POSLATAM - Buzios
167
10/22/2012
V POSLATAM - Buzios
168
10/22/2012
V POSLATAM - Buzios
169
10/22/2012
V POSLATAM - Buzios
170
Sistemas criogênicos Soprador de N2g • a partir de N2L • compressão de N2g do ar
10/22/2012
V POSLATAM - Buzios
171
10/22/2012
V POSLATAM - Buzios
172
28
10/22/2012
Sistemas criogênicos
10/22/2012
V POSLATAM - Buzios
Sistemas criogênicos
173
Crio vs RT
10/22/2012
V POSLATAM - Buzios
174
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)
10/22/2012
V POSLATAM - Buzios
175
10/22/2012
V POSLATAM - Buzios
176
10/22/2012
V POSLATAM - Buzios
177
10/22/2012
V POSLATAM - Buzios
178
29
10/22/2012
10/22/2012
V POSLATAM - Buzios
179
10/22/2012
V POSLATAM - Buzios
180
10/22/2012
V POSLATAM - Buzios
181
10/22/2012
V POSLATAM - Buzios
182
10/22/2012
V POSLATAM - Buzios
183
10/22/2012
V POSLATAM - Buzios
184
30
10/22/2012
Coleta – fontes de raios-X • • • •
Anodo Tubo selado Sincrotron Fita adesiva (http://www.youtube.com/watch?v=LQBjR F9mX1Y) • Coleta remota / in situ 10/22/2012
V POSLATAM - Buzios
185
10/22/2012
V POSLATAM - Buzios
186
LNLS
INMETRO
Cu
Mo
Diffraction directly from cry stallization screening plates
Single cry stal diffraction
10/22/2012
V POSLATAM - Buzios
Dual wavelength
187webs ite Images from Agilent
10/22/2012
V POSLATAM - Buzios
188
anodo rotatório
… o rder a d i f fracto meter
10/22/2012
V POSLATAM - Buzios
189
10/22/2012
V POSLATAM - Buzios
190
31
10/22/2012
10/22/2012
V POSLATAM - Buzios
191
10/22/2012
V POSLATAM - Buzios
192
10/22/2012
V POSLATAM - Buzios
193
10/22/2012
V POSLATAM - Buzios
194
10/22/2012
V POSLATAM - Buzios
195
10/22/2012
V POSLATAM - Buzios
196
32
10/22/2012
Geradores: Funciomanento • Tubo Selado • Anodo rotatório • Sincrotron
10/22/2012
V POSLATAM - Buzios
10/22/2012
V POSLATAM - Buzios
10/22/2012
V POSLATAM - Buzios
197
199 Intr Macr Xt, McPherson
ITC,201F
10/22/2012
V POSLATAM - Buzios
198
10/22/2012
V POSLATAM - Buzios
Princ Ptn x-ray diffr, Drenth, 3r d Ed200
10/22/2012
V POSLATAM - Buzios
Princ Ptn x-ray diffr, Drenth, 3r d Ed202
•© 2006 •Academic Press
33
10/22/2012
10/22/2012
V POSLATAM - Buzios
Princ Ptn x-ray diffr, Drenth, 3r d Ed203
10/22/2012
V POSLATAM - Buzios
204 Intr Macr Xt, McPherson
Rigaku • • • •
10/22/2012
V POSLATAM - Buzios
205
•© 2006 •Academic Press
Anodo rotatório Tubo selado Ambos, opções Cu, Mo Detector RaxisIV (Rigaku) ou Mar – IP / CCD
10/22/2012
V POSLATAM - Buzios
206
Bruker - AXS • Tubo selado • Detectores: CCD • Fontes: Cu / M o
10/22/2012
V POSLATAM - Buzios
207 Intr Macr Xt, McPherson
10/22/2012
V POSLATAM - Buzios
208
34
10/22/2012
Agilent (Oxford Diffraction)
Bruker - AXS
• Tubo selado • Detectores: CCD • Fontes: Cu / Mo
10/22/2012
V POSLATAM - Buzios
209
Agilent (Oxford Diffraction)
10/22/2012
V POSLATAM - Buzios
10/22/2012
V POSLATAM - Buzios
210
Agilent (Oxford Diffraction)
211
10/22/2012
V POSLATAM - Buzios
212
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 10/22/2012
V POSLATAM - Buzios
213
10/22/2012
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103 -97331997000400002
V POSLATAM - Buzios
214
Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997
35
10/22/2012
Brazilian Sy nchrotron Light Laboratory (LNLS) Campinas - Brazil 10/22/2012
V POSLATAM - Buzios
215
10/22/2012 ITC, F
V POSLATAM - Buzios
216
17 Fev 2004
(ainda um pouco oscilante…)
Typical time dependence of electron current in the LNLS storage ring (April 1997).
10/22/2012
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103 -97331997000400002
V POSLATAM - Buzios
217
10/22/2012
V POSLATAM - Buzios
218
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. 10/22/2012
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103 -97331997000400002
V POSLATAM - Buzios
219
Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997
10/22/2012
http://www.scielo.br/scielo.php?pid=S1516-14392002000100002&script=sci_arttext V POSLATAM - Buzios 220 Mat. Res. vol.5 no.1 São Carlos Jan./Mar. 2002
36
10/22/2012
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. 10/22/2012
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103 -97331997000400002
V POSLATAM - Buzios
221
Location of the nine beamlines to be opened to users in 1997 (seven) and 1998 (two). 10/22/2012
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103 -97331997000400002
V POSLATAM - Buzios
Braz. J. Phys. vol.27 n.4 São Paulo Dec. 1997
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
10/22/2012
V POSLATAM - Buzios
223 © 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).
10/22/2012
V POSLATAM - Buzios
224
10/22/2012
V POSLATAM - Buzios
226
SAXS – SAS1 • • • • •
Primeira linha do LNLS Agora em outra saída do anel Novo detector – 2D Mesmo desenho da MX1 Detalhes da linha antiga:
10/22/2012
V POSLATAM - Buzios
225
37
10/22/2012
SAXS – SAS1
10/22/2012
10/22/2012
http://www.scielo.br/scielo.php?pid=S1516-14392002000100002&script=sci_arttext V POSLATAM - Buzios 227 Mat. Res. vol.5 no.1 São Carlos Jan./Mar. 2002
V POSLATAM - Buzios
229
10/22/2012
V POSLATAM - Buzios
228 L. A. Bernardes ©
10/22/2012
V POSLATAM - Buzios
L. A. Bernardes ©
230 L. A. Bernardes ©
Detectores: Funcionamento • Placa de imagem • CCD
10/22/2012
V POSLATAM - Buzios
231
10/22/2012
V POSLATAM - Buzios
232
L. A. Bernardes ©
38
10/22/2012
Rigaku Ultra18X (IFSC, LNLS) Image Plate Mar345dtb
10/22/2012
V POSLATAM - Buzios
IP - M ar345dtb
233
10/22/2012
V POSLATAM - Buzios
235
10/22/2012
V POSLATAM - Buzios
V POSLATAM - Buzios
236
M X1 – M arCCD
M ardtb
10/22/2012
234
IP – R-AXIS V
IP - M ar345dtb
10/22/2012
V POSLATAM - Buzios
237
10/22/2012
V POSLATAM - Buzios
238
39
10/22/2012
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. 10/22/2012
V POSLATAM - Buzios
239
•© 2006 •Academic Press
10/22/2012
V POSLATAM - Buzios
240
http://www.mar-usa.com/support/downloads/sx_series.pdf
M X2 - M arMosaic
10/22/2012
V POSLATAM - Buzios
241
http://www.marresearch.com/marccd.ht m
10/22/2012
M X2 - M arMosaic
10/22/2012
V POSLATAM -http://www.marresearch.com/products.mx-series.html Buzios 243
V POSLATAM - Buzios
242
Quantum
10/22/2012
V POSLATAM - Buzios
244
40
10/22/2012
10/22/2012
V POSLATAM - Buzios
245
10/22/2012
V POSLATAM - Buzios
246
10/22/2012
V POSLATAM - Buzios
•© 2006 •Academic Press
M esa detectora
10/22/2012
V POSLATAM - Buzios
247
10/22/2012
V POSLATAM - Buzios
•© 2006 •Academic Press
248
249
41
View more...
Comments
