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Growing Protein Crystals. Using Calcium-Integrin Binding Protein as a Model Presented by Chad Blamey. FBP www.scripps.edu/~arvai/ xtals/xtals.html. Goals. What are good crystals Why getting good crystals is important Understand how crystals grow

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growing protein crystals

Growing Protein Crystals

Using Calcium-Integrin Binding Protein as a ModelPresented by

Chad Blamey

FBP

www.scripps.edu/~arvai/ xtals/xtals.html

goals
Goals
  • What are good crystals
    • Why getting good crystals is important
  • Understand how crystals grow
  • Discus techniques for crystallizing proteins
    • Application type of discussion
  • Strategies for optimizing crystal growth
    • Understanding your favorite protein
  • CIB as a model
    • My favorite protein!!!
  • Lysozyme demonstration
lysozyme demonstration

2l

2l

4l

Lysozyme Demonstration

Buffer

30% w/v Polyethelene glycol 5000

1 M NaCl

50 mM NaAcetate pH 4.5

Lysozyme Protein

100mg/ml

50 mM Na Acetate pH 4.5

glass slide

Should make large crystals in less than 15 minutes.

We will watch it for the hour of the lecture.

everyone should know
Everyone Should Know
  • Protein crystals are precipitated protein in solutions
  • You can think of them highly concentrated aqueous solutions (usually about 500 mg/ml)
  • Amorphous precipitation is random
  • Crystals are ordered
    • This is the property we are interested in
    • Figure 3.1 CMCC
    • Gray areas!

Crystals

Precipitation

Crystalline

crystallization needs
Crystallization: Needs
  • Obtaining quality crystals is by far the limiting step to solving a structure
  • Crystals need to be of sufficient size and quality to diffract x-rays
  • Size: Normally should be 100 m in smallest dimension
  • Quality: Reflections collected from diffraction data are the primary source of data to build an electron density map, therefore quality of protein model depends greatly on crystal quality
    • Growing good crystals is key to a good structure
crystallization
Crystallization
  • With enzymes is is often important to maintain enzymatic activity in crystal
    • Some enzymes can function in crystal
  • Best way to test crystal quality is by mounting a crystal and attempting to diffract x-rays
    • Visual inspection helpful too
    • May not be meaningful
low vs high data
Low vs High Data
  • Difference between 9.0 Å and 4.5 Å
  • The higher the resolution the better!
  • CIB crystal spots

9 Å

4.5 Å

good vs poor data
Good vs Poor Data

Ca+007

M035

4.5 Å

Poor, smeary spots

Notice ‘twined’ spots

4.1 Å

Good! Round spots

Higher order visible (circle)

spot prediction
Spot Prediction

Crystal M035

Do spots match mathematical predictions?

how do proteins crystallize
How Do Proteins Crystallize?
  • For crystallization to occur it has to be thermodynamically favorable
  • Precipitants remove available water forcing proteins to associate with each other
  • Hopefully in a organized fashion

Water

+

+

Protein

+

precipitant

+

-

-

-

-

polyethelyene glycol

salts

sugars

organic solvents

growing crystals hanging drop method

[X]

[Y]

Growing Crystals: Hanging Drop Method

crystals

  • Widely used
  • Vapor diffusion
  • Drop equalizes with reservoir
  • Volume of drop slowly decreases
  • Protein concentration slowly increases
  • CMCC Figure 3.2

drop

reservoir

Sitting drop

phases of proteins in solution

Crystals

Figure 3.3

Precipitation

Metastabile

Supersaturation

Growth & Nucleation

Solubility

Barrier of Nucleation

Undersaturated

Growth only

[Protein]

Soluble protein

Phases of Proteins In Solution

Not to be confused with phases of light

CMCC figure 3.3

nucleation growth

Phase diagram

Figure 3.3

Metastabile

Supersaturation

Solubility

Undersaturated

[Protein]

Nucleation & Growth

Basic concept:

  • Concentrate solution enough so nucleation occurs in only a few cases
  • Initial growth pulls some protein out of solution
  • Reducing [protein] back into metastable range
  • Grow only a few large crystals
optimize crystal growth

[X]

[Y]

Optimize Crystal Growth
  • The number of factors can be overwhelming
  • Focus on those factors which most effect growth
  • Set up arrays to vary two different conditions at once
  • Cross your fingers
the tricky part
The Tricky Part
  • Conditions for crystallization are dependent on each-other
  • Crystal quality will change as you vary growth conditions
  • Figure 3.4

[B]

[A]

For solution made up of three parts A, B and C.

Changing [C] will effect the quality of the crystal in

terms of [A].

growing crystals other techniques

Spin

6 hours

12 hours

0 hours

Growing Crystals:Other Techniques

Ulatacentrifugation

  • Spin at extremely high speeds, hundreds of thousands of g’s
  • Slowly increases the relative protein concentration

Dialysis

  • Uses liquid-liquid diffusion
  • Diffusion is slow
  • Rate controlled by membrane
crystal screens
Crystal Screens
  • Hampton Research screen tests a wide assortment of conditions of salts, buffers, pH’s and additives
    • Best conditions from literature
  • Often first hits with screens are small poor quality crystals
  • Do not use the absence of crystals as a gauge of conditions rather use solubility
factors effecting crystal growth
Ionic Strength*

Specific Ions (Ca2+)

Protein Concentration*

Detergents

Inorganic Precipitant

pH*

Temperature*

Time

Monodispersion*

Vibrations

Pressure

Gravity

Relative Proportion of Conditions

Purity Of Protein*

Access to water*

Ligands

Binding partners

Factors Effecting Crystal Growth

*Most important

characterization of protein
Characterization of Protein
  • Of course the more you know about your protein the easier it is to manipulate
  • Cystine is often the most critical a.a.
    • CIB has three and no disulfide bonds, but cause multimers
  • Key ligands and metals, like Ca (for CIB)
  • Stability in certain solutions
    • Hydrodynamic radius (NMR)
    • Stability (CD is great)
    • Dynamic light scattering
    • Mass spec
cib protein characterization

CIB purified w/ reducing agents

CIB purified w/o reducing agents

IEF

Western-

Blot

IEF

SDS-PAGE

Marker

No DTT

DTT

CIB

pH 5.6

22

CIB Protein Characterization

Further characterization of a protein can

improve purity and therefore crystal quality

ligands and co crystallization
Ligands and Co-crystallization
  • Try to obtain crystals with different ligands and/or co-crystallize with another protein
    • Metals, peptides & binding proteins
    • Proteins with a known structure can simplify the process
  • Enzymes and Substrate complexes
    • Non-competitive inhibitors
    • Substrate analogs
  • Often changes protein conformation
    • Two structures!
    • This gives information about how the protein function
additives
Additives
  • Often designed to reduce strength of protein-protein interaction
  • Detergents important category
  • Reducing agents
  • Organic solvents
small crystals
Small Crystals
  • Often small crystals can be made larger by microseeding new drops with previously grown crystals or adding more protein solution
  • Multinucleation can be avoided by reducing the temperature or adding glycerol
  • Crystals only need to be large enough to diffract x-rays well
radical approaches
Radical Approaches
  • Remove either N- or C-terminus by weak proteolysis or by molecular cloning
    • Often termini can be disordered which interferes with lattice formation
  • Crystallize with a fusion protein
    • Fusion proteins are well documented with a solved structure that easily from a lattice, example: GST
    • “Pull” the fusion protein into an ordered crystal
    • Can use the protein for molecular replacement to solve phase
    • Many recombinant proteins are purified using fusions anyways, i.e. not hard to try
radical approaches cont
Radical Approaches, Cont
  • Mutants:
    • Specific residues problem residues can be mutated using recombinant DNA technology
    • Domains can be crystallized separately
  • Issues:
    • Different conformations from the native state likely
    • Domains can only be part of the story
    • Changing the means starting over in terms of crystallization solution
discussion
Discussion
  • Given a hypothetical protein that doesn’t give you any positive hits in your first screen what could you do to obtain quality crystals?
    • Meaning: almost no precipitation in each drop!
  • Likewise what if you get lots of precipitation?
  • Say on the other hand at room temperature you have a condition with lots of tiny crystals, what can be done to reduce the amount of nucleation?
lysozyme
Lysozyme
  • Vapor diffusion takes about 12 hours to complete. Where on phase diagram did the [lysozyme] start given almost no vapor diffusion occurred?
  • Lysozyme in other solutions crystallizes much more slowly (period of days). Which solutions would yield higher quality crystals? Why?
  • What are some of the properties of lysozyme that allow it to crystallize so quickly?
slide28
CIB
  • Similar to a ubiquitous protein Calmodulin
  • Binds calcium
  • Regulates other proteins (13 so far)
  • Found in most tissues types: brain, muscle etc.
  • No enzymatic activity
cib discussion
CIB Discussion
  • What are some possible techniques that could be used to obtain CIB crystals?
  • What about the cystines of CIB?
  • Why is it important that the radius of CIB is smaller when it is bound to calcium?
  • CIB contains a surface exposed hydrophobic patch, how could this information change your crystallization conditions?
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