Protein Crystallography
At SSRL
By: Silpa Nalam and Andrea Kurtz
Advisors: Aina Cohen, Paul Ellis, and
Nick Sauter
Part 1: Introduction
Project Description
•Protein Crystallization
•Web Page Design
Background on X-rays
• Advantages of SSRL
x-ray beam lines over
home x-ray tubes
• higher intensity
• tuning the x-rays allows one to
choose one wavelength
• better resolution
Diffraction
Pattern
How is a diffraction
pattern made?
• The pattern is made from
diffracted x-rays by the atoms
of the sample
• math and computers are used
for interpretation because xrays cannot be focused by a
lens system
• dimensions of spots give unit
cell information and their
intensity gives the arrangement
of the atoms in the unit cell
What is a Protein?
•Basic Structure
•polypeptide chain and its average
size is 10,000 Daltons
•R groups have 20 different
combinations and help the protein
bind to ligands, catalyze chemical
reactions and direct the polypeptide
in its conformation
•Functions
•enzymes, structural elements,
antibodies, hormones, and electron
carriers
Protein Structure
• 4 Levels of structure
• give the protein a 3-D shape
which helps carry out its
functions
• primary=sequence of R groups
• secondary=random coil, alpha,
or beta structures
• tertiary=folding of secondary
structures (biologically active)
• quaternary=more than one
polypeptide chain
• Protein sensitivity
•
denaturation (protein unfolding)
caused by heat, chemicals,
changes in pH
Alpha Helix
Beta Sheet
Common Proteins in the Body
Myoglobin is used as an oxygen
storage molecule that can be
found in the muscle tissue of
vertebrates
Hemoglobin is a unique blood
protein that carries oxygen from
the lungs to the rest of the body.
Leghemoglobin is a type of
hemoglobin found in plants. It is
an important part of the nitrate
fixation process
Crystallization with X-rays
• Usefulness of x-rays
• wavelength of x-rays is 10-8 cm
• Difficulties in Crystallization
• not usually in a crystalline fashion
• not suited to be in crystalline form
• protein has an extreme sensitivity
• Why Crystallization?
• provides the answer to many structure related questions
Background on Crystals
• A crystal is a periodic
arrangement of unit cells
in a lattice. The unit cell
is repeated in a 3-D lattice
to make a crystal.
• Common example of a
crystal in a lattice is
sodium chloride
Materials for Experiment
Linbro Plate
Myglobin
24 well tray and
each well is 1.5
cm in width and
1.8 cm in depth
The protein is placed
in a reagent that
reduces its solubility
to close to
precipitation. The
concentration should
be 5-50 mg/mL
Cryo Solutions
Allows for the
crystals to grow.
They can be
bought or
homemade.
Gel
The gel is
used to
ensure the
coverslips
stay over
the well
Procedure For Experiment
Vapor-Diffusion Method
• The protein/precipitant mixture
in the drop is less concentrated
than in the well solution.
• Water evaporates from the drop
until the concentration of the
precipitant in the drop is the
same as in the well.
• The conditions in the drop
determine whether the protein
will crystallize or not. Such as:
ionic strength, temperature, and
protein concentration.
Finished Tray
Variables
• As the precipitant point of the protein is approached, factors such as
pH, temperature, ionic strength, and choice of buffer control whether
the protein will separate from the solution as a crystal or a precipitate.
• The pH, type of cryoprotectant (PEG or MPD) and buffer should be
varied to get the protein in crystalline fashion.
• It is hard to crystallize proteins under normal conditions because the
crystal might react with water vapor.
The crystals are usually
grown in a cold room.
Key Points
•
•
•
•
Reproducibility
Crystallography: art or science?
Software based trials
Crystallization process
• growing crystals
• analysis of crystals
• production of data
• conclusion
• theory of crystallization
Protein Crystallography at SSRL
Part II: Determining Crystal Quality
and
Data Collection and Analysis
Background on Crystals
• A body that is formed by the
solidification of a chemical
element, a compound, or a mixture
and has a regularly repeating
internal arrangement of its atoms
and often external plane
faces
7 Classes of Unit Cells
• Described by three axes (a,b,c)
and three angles (,,)
230 Space Groups
• Characterized by symmetry elements
such as rotational and screw axes
The presence of symmetry elements
simplifies data collection and analysis.
Left: Single molecule of bovine pancreatic
trypsin inhibitor (BPTI), considered the
asymmetric unit in the unit cell below
Below: Packing diagram for orthorhombic unit cell of
BPTI in the most common space group, P212121
Macromolecular Crystallization
Crystallization of the protein is only the first step…
Examination of Samples
Protein remains soluble
Crystalline precipitate
Amorphous precipitate
Gel precipitate
Cyclophilin crystals
Crystal Quality at a Glance
Characteristics of Crystals Suitable for Analysis:
• Large size, i.e., average length of approximately 200m
• Single crystals with smooth, distinct faces and edges
• Color (in many cases) or opacity (for myoglobin)
Common Protein Crystal Defects:
• Bunching or twinning
• Size deficiencies
• Denaturation with age
Mounting and Flash-Cooling
Many protein crystals are extremely sensitive
to changes in temperature and surrounding
conditions (i.e., presence of original solvent).
The crystal must be flash-cooled to 100K in a
stream of liquid nitrogen to prevent damage
from high-intensity x-rays.
Teasing the crystal from the coverslip droplet
Crystals grown in the
cold room must be
flash-cooled and
transported to the
experimental hutch
with cryo-tools.
Copper pin on goniometer head in liquid nitrogen stream
Data Collection Variables
According to the size of the crystal
and the type of protein, the following
factors are assigned prior to data
collection:
• X-ray beam wavelength (on tuneable beam lines)
• Crystal-to-detector distance
•  = degree of oscillation around phi axis
• Exposure time
• Start and end -angle
Sample Control Panel at Beam Line 9-1
The Diffraction Pattern
Key Features:
• spot separation
• unit cell dimensions
• spot intensity
• unit cell contents
Myoglobin diffraction pattern at 1.598Å resolution
Left: Bragg’s Law and reflection of x-ray beams from crystal planes
Origins of the Diffraction Pattern
Data Interpretation
Using Mosflm, we can perform the following functions
on the data embedded in the diffraction pattern:
• Autoindexing and Cell Refinement
• unit cell determination
• mosaicity
• orientation matrix
• Integration
• intensity: count-integrated and profile-fitted on each image
• combine reflections on multiple images
• Scaling and Merging
• combining equivalent reflections
• crystal breakdown and beam decay
Tests of Data Quality:
• R-factor
• Percentage of data measured
• Systematic absences
• I/I and F/F
The importance of resolution
in structural determination
Methods of Solving Structures
• Direct - small molecules
(less than 100 amino acids)
with very high resolution data
• Multiple Isomorphous
Replacement (MIR)
• Multiwavelength Anomalous
Dispersion (MAD)
• Molecular Replacement
Method (MRM)
Random Amplitudes
Random Phases
Above: Fourier synthesis diagrams. The red lines represent the
model that was used to find the actual amplitudes and phases. Both
factors are essential, since phases are not directly measurable and
must be calculated from amplitudes
Future Improvements
• Automated Batch Handling and X-Ray Diffraction Analysis
• Integration of Computer Technology with Human Ability
• Increased Internet-Based Interfacing for Data Collection,
Analysis, Storage, and Retrieval
Crystallographer’s Creed
This is my x-ray machine. There are many like it, but this one is mine. My x-ray
machine is my best friend. It is my life. I must master it as I must master my life.
Without me, my x-ray machine is useless. Without my x-ray machine, I am useless. I
must collect my data true. I must phase faster than my enemy who is trying to publish
before me. I must shake him before he bakes me.
I will.
-Dr. Bernhard Rupp, LLNL