ELECTRON

DENSITY MAPS

- BUSHRA
NOOR
BMS 3rd YR.

The three-dimensional
arrangement of electrons
within the molecule is
represented as an Electron
Density Map.

Powerful computer programs are used

for the calculations of electron density
maps.

1.
The individual
intensity of each
spot

3.
2.
The phase of the
The overall
X-ray waves
pattern of spots
corresponding to
obtained
each spot

(i)
INDIVIDUAL
INTENSITY OF
EACH SPOT

Intensities, Ihkl , are directly measured by the detector or

recorded on photographic film.
o But in order to calculate an electron density map the intensity
of each Bragg reflection, Ihkl , is converted into a structure
factor, Fhkl.
Therefore, intensity is converted into structure factor using the
following relationship:

|Fhkl |2 = K Io / LP

Here,
K is scaling factor, dependent on properties such as beam intensity &
crystal size.
L is Lorentz factor
P is polarization factor, which corrects for differences experienced by
electric components of the X-ray wave parallel to the lattice plane (hkl)
compared to those perpendicular to the plane

 An observed estimate of the structure factor, Fhkl obs, for

each Bragg reflection is made
Once a model of the structure has been proposed, a
process called refinement is employed in which there is a
repetition of a series of computer programs.
Each of this generates a calculated estimate of the
structure factor, Fhkl calc, based on the current model.

The difference between these two, Fhkl ,

is given by-

Fhkl = Fhkl obs Fhkl calc

To know about the quality of the final structure and indication

of how acceptable it is, R factor is calculated.

* R - FACTOR
The R factor represents a summation of all the structure factors
for the proposed structure giving an :

R = |Fhkl | / |Fhkl |obs

Lower the R-factor, finer is the structure

A good structure have a R-factor of 15-20%

(ii)
THE OVERALL SPOT
PATTERN

 Computer programs are there to give an estimate of the

lattice constants and the space group of the structure.
It helps in analyzing the symmetry and relative intensities of
spots observed within the overall pattern of diffraction.
We can also estimate the number of asymmetric units in the
unit cell and the volume of each from this; which in turn helps
us obtaining the number of sample molecules in the asymmetric
unit.

(iii)
THE PHASE OF X-RAY
WAVES

To describe the diffracted x-ray waves and hence, determine the

three-dimensional location of the diffracting atom we need to
know about the 1. Amplitude
2. Wavelength
3. Phase angle, hkl

Because each Bragg reflection results from a

combination of wavelets scattered by all the atoms in the
molecule, different reflections have different phase
angles which must be determined in order for an electron
density map to be calculated.

Phase determined

Experimental data
collected

Model built over it

Refinement of the
model comparing
with empirical data

Optimised protein structure

But phase angles cannot be obtained directly from

intensities of Bragg reflections detected in the
diffraction pattern. That is, the diffraction data-set
from a crystal contains the amplitude information but
has lost phase information.

 Geometric
description of
diffracted beams
for different hkl.
Three beams are
shown which are
out of phase and
have different
phase angles.
The structure
factor Fhkl is a
resultant vector
between the
cosine and sine
vectors A and
B . The angle
formed between

 From the representation, it can be considered that |Fhkl | is

the resultant vector between two vectors A hkl and B hkl
representing cosine and sine waves, respectively.
The intensity is also geometrically related to their magnitude
as

Ihkl = |A hkl |2 + |B hkl|2 = |Fhkl |2

Structure factors, Fhkl may directly be obtained
The phase angle is equal to the ratio of these vectors:

= tan hkl = B hkl / A hkl

Phase angle, can not be directly obtained

 Since we need to know both the phase and the amplitude of

each Bragg reflection to calculate the electron density map,
this lack of phase information provides a barrier to structure
calculation called the phase problem.
A number of strategies have been developed for the recovery
of phase information

ISOMORPHOUS
REPLACEMENT

MOLECULAR
REPLACEMENT

ANOMALOUS
SCATERRING

1. ISOMORPHOUS REPLACEMENT
This technique was developed for proteins by Kendrew
and Perutz.
It involves the introduction of a heavy metal atom such
as mercury, platinum, uranium etc. at a unique location
within the molecule called single isomorphous
replacement.
This is achieved by soaking a crystal with a solution of a
reactive chemical containing the heavy metal which
then attaches to the biomacromolecule.

 When X-rays are diffracted through this crystal

containing the heavy metal, the location and phase of
Bragg reflections from metal atoms can be identified by
comparison with a data-set from the crystal lacking heavy
metal.

It is important that this reaction does not result

in an altered structure, crystal packing or unit
cell size

The structure factors for native protein (Fhkl P), heavy-metal

modified protein (Fhkl PH) and heavy metal alone (Fhkl H), are
related to each other as vector quantities as:

Fhkl H = Fhkl PH - Fhkl P

Structure factor vectors forNative protein = Fp

Heavy-metal modified protein
= FPH
Heavy metal alone = FH
with the corresponding
relative phase angles (P and
PH)

Now, the location of these heavy

metals inside crystal lattices are
estimated by using difference
Patterson maps.
Here, if two atoms in a unit cell
are separated by a vector (uvw),
there will be a peak in the
Patterson map at (uvw).
Therefore, proteins that are
modified with heavy metals are
dominated by vectors between
the metal and other atoms.

P(uvw) = 1 / V |Fhkl |H2 cos 2(hu + kv + lw)

The location of the heavy metal is calculated using the above
equation.

Therefore ,
As we now know the three dimensional locations of the heavy
metal (i.e. both hkl H and |Fhkl |H), |Fhkl |P and |Fhkl |PH
(experimentally measured from diffraction intensities), the
relative phase angle for the protein, hkl P, can be calculated
from the following equation:

hkl P = hkl H cos1 [(|Fhkl |PH2 |Fhkl |P |Fhkl |H) /

2|Fhkl |P|Fhkl |H]

 But, two relative phase angles are

generated in case of these proteins,
one of which is true and the other
imaginary.

Therefore, in order to distinguish which of the pair is true, a

second (and sometimes a third) heavy metal at another unique
location in the unit cell is included.
This experiment is called multiple isomorphous replacement.

It results in a single value for hkl P

The effect of this may be seen using a geometrical construction
called the Harker construction.

The Harker
construction
(a) Single isomorphous
replacement
(b) Multiple
isomorphous
replacement

2. MOLECULAR REPLACEMENT
In this, a pair of related molecules are taken, for which the structure
of one is known - the Model, and the other unknown molecule the
Target.

1st stage
1. The model is rotated around a set of three angles relative to the
observed structure factors of the data-set from the target.
2. For each orientation structure factors are calculated from the model.
3. The agreement between the calculated structure factors and the
observed values from the diffraction experiment is then used to
identify the orientation of the known molecule that most closely
matches that of the unknown molecule in the crystal.

The mathematical tools to carry out these operations are known as

rotation functions.

 2nd stage
1. This stage is achieved with a translation function
2. The target is moved relative to that for the model by
translation but without changing their relative orientation.
3. Again the agreement of the structure factors are used to
identify the correct translation. If the correct orientation and
translation can be identified, then the model may finally be
used to calculate phases for all the structure factors.
4. Electron density maps may then be calculated using phases
from the model structure and weighted magnitudes from the
unknown structure.
5. The resulting map may be examined to determine the
unknown structure.

GENERATED
ELECTRON
DENSITY
MAP

ACTUAL
MODEL
PROPOSED
MODEL

!
U
O
Y
K
N
A
H
T