Study Questions and Answers
th
October 28
1.
, 2008
Synchrotron Trip:
Discuss the advantages of measuring data at a Synchrotron as compared to an in-house home
source.
Pro:
•
tunable wave-length: Solving the phase problem with MAD/ MIR, etc.
•
greater intensites often results in better resolution
•
faster data collection (10min vs. several hours)
Contra:
•
Radiation dammage: High dosage can break bonds, creates free radicals that can ruin the
crystal lattice.
•
Availability: beam time must be applied for, and in-house machines can usually be used for
as long as necessary to complete the data
2.
CryoCrystals:
X-ray data are usually collected while the crystal is immersed in a stream of liquid nitrogen. This
reduces the amount of radiation dammage signicicantly.
Suppose your crystal grew in a solution
of 20 mM Tris-buer, pH 8.5 and 200 mM Ammonium Sulphate (in addition to you protein).
What would happen if you held this crystal directly into the stream of liquid nitrogen and how
can you prevent that?
Eect:
Under these conditions, the water in solution would crystallise itself. The crystal lattice
of ice would destroy the crystal lattice of the protein, rendering the crystal useless.
Cure:
Addition of
Typical cryo-protectants and required concentrations:
•
glycerol (20-30%)
•
sugars (glucose, sucrose, 25-35%)
•
Polyethylene Glycols with low average molecular weight (400-1000Da; 20-35%)
• LiCl
3.
cryo protectant.
(2M)
Crystal Morphology
Assign the following four shapes, as seen through a microscope, to
(a) a drilling (three crystals grown together)
(b) an amorphous non-diracting aggregate
(c) a crystal with a cubic crystal form
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(d) a bunch of small crystals grown together
Answers:
aC: no edges at all
bB: looks like a cube seen from the side
cA: a monocrystal cannot have inward corners, i.e. angles
> 180◦
dD: this looks pretty, but is not useful because it is not a monocrystal
4.
Supermicroscope:
Someone suggests to build a light microscope with such a good magnication that we can see single
atoms. With such a microscope at hand, we could look at proteins without the need of crystal
and without the need of an X-ray experiment. Why can such a microscope not be built?
Answer:
Light can only separate objects which are more than half its wavelength apart. This
is hidden in Bragg's Law, where
dmax = λ/2
θ = 90◦ (i.e. a reection that comes back
> 400nm(4000Å) so one can only separate
for
towards the beam). Visual light has a wavelength
of
objects which are more than
200nm = 20Å
apart.
Atoms, however, are about 1.5-1.7Å apart and even secondary structure elements are a lot smaller
than 200nm. Therefore molecules are too small for visible light.
Swing-out detectors:
At synchrotrons one can often only move the detector in one direction, back and forth along the
beam. More sophisticated setups like the Smart 6000 allow to move the detector back and forth
Synchrotron
home source
detector
translation
beam
beam
detector
translation
and around the crystal.
What is the advantage of the latter setup?
Answer:
tor
tec
de ation
rot
5.
There are two advantages to the second setup.
2
The rst is (again . . . ) resolution. A typical detector with a radius of 1020 cm cannot be moved
closer than 10cm to the crystal. With the synchrotron setup this limits the maximal angle 2∗θmax
◦
◦
to about 45 , i.e. θmax = 22.5 . At a typical wavelength of 1Å, this results in a maximal resolution
of
λ
2 ∗ sin(θmax )
1Å
=
2 ∗ sin(22.5◦ )
= 1.3Å
dmax =
(1)
For some (rather rare) cases this is not enough. If the detector can be rotated, this problem is
overcome.
spot
overlap
good spot
separation
high resolution
splot caught by
rotated detector
The second is the separation of the spots.
short distance
long crystal−detector distance
With a rotatable detector one can move the detector back and hence separate the reections and
still collect the highresolution reections by rotating the detector.
6.
Improving Data Quality:
Which of the following factors tend to help in determining a crystal structure (give reasons)?
(a) the presence of metal atoms such as iron
Answer:
can be used for phasing (MAD, SIR, . . . )
(b) a high sequence similarity with a protein of known structure
Answer:
can be used for molecular replacement
(c) aromatic residues at the N and/or C-termini
Answer:
not directly related to crystallography, but might lower solubility.
(d) data collected at 100 K
Answer:
gives better data, reduces radiation damage
3
(e) high solvent content
Answer:
increases data to parameter ratio, since number of reections depends on size of
unit cell, not its content. On the other hand it can lower the stability of the crystal and result
in increased thermal motion factors which weakens the signal.
7.
Symmetry related reections:
One of the symmetry operators in the spacegroup
I21 3
(the space group of e.g. cubic Insulin), is
represented by the following matrix:


0 0 −1


 −1 0 0 
0 1 0
This means that any two reections



h


 k 
l





0 0 −1
h
−l


 


 −1 0 0  ×  k  =  −h 
0 1 0
l
k
and
are identical.
An integration program reports the following three reections and their intensities:
h
k
l
intensity
2
4
0
257
0
-2
4
120
-4
0
-2
265
Why should you be suspicious about this result?
Answer:
the three reections are symmetry related:






0
2
0 0 −1




 
 −1 0 0  ×  4  =  −2 
4
0
0 1 0





and

0 0 −1
0
−4




 
 −1 0 0  ×  −2  =  0 
0 1 0
4
−2
(2)
Therefore their intensities must be equal, but not dierent by a factor of two (NB: In a real
experiment such deviations may yet occur.
The second reection (0 -2 4) would probably be
marked as an outlier and rejected from the data set).
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