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Growing Protein Crystals. Using Calcium-Integrin Binding Protein as a Model Presented by Chad Blamey. FBP 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 xtals/xtals.html

  • 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




Lysozyme Demonstration


30% w/v Polyethelene glycol 5000

1 M NaCl

50 mM NaAcetate pH 4.5

Lysozyme Protein


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!




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
  • 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



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












polyethelyene glycol



organic solvents

growing crystals hanging drop method



Growing Crystals: Hanging Drop Method


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



Sitting drop

phases of proteins in solution


Figure 3.3




Growth & Nucleation


Barrier of Nucleation


Growth only


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






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



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



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


6 hours

12 hours

0 hours

Growing Crystals:Other Techniques


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


  • 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*


Inorganic Precipitant








Relative Proportion of Conditions

Purity Of Protein*

Access to water*


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










pH 5.6


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
  • 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
  • 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?
  • 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?
  • 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?