Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

Structure Determination Notes:
 Dimentions in Life Sciences:


 Ways to visualize proteins:

 X-ray diffraction has
the highest resolution
 Electron Microscopy
has the widest range

 Resolution vs Noise
 Resolution = quantitative measure of the level of detail that can be discerned in an
image of an object
...
Smaller numbers are
better!
 Noise = random degradation of an image due to local fluctuations in the measured intensity at
individual coordinates
...




better than light microscope because light can damage your
sample while electrons cannot!!!

 Specimen Preparation:


Negative Stain: the background is stained, leaving the actual specimen untouched, and thus
visible
...
This techniques creates a three dimensional
view of the specimen with the specmen itself appearing darker
...
The cold fractured surface is then shadowed with evaporated platinum or gold in a
high vacuum evaporator
...
The
thin layer of tissue remains bound to the metal replica so it can be immunogold labeled with
antibodies to the structures of choice
...
An aqueous sample solution is
applied to grid-mash and plunge-freeze in liquid ethane
...
These techniques are based on
observing the scattered intensity of an X-ray beam
hitting a sample as a function of incident and
scattered angle, polarization, and wavelength or
energy
...
Overproduce and Concentrate Protein
2
...
Optimize successful ones (pH, salt, ligands, etc)

Where do we get the
phases from?


Collect Data
4
...
Collect native data (usually at a synchroton
10,000 brighter
than home)
6
...
Solve heavy atom positions using Patterson methods
8
...
From the “leftovers”, calculate protein phases (1 MAD or > 2 heavy atom
derivative)
10
...

12
...

14
...
Minimize structure against the data by calculating maps phased with the
structure (automated)
16
...
Repeat 15 and 16 until Rfactor converges



pertub the
system:
 MIR
(position of a
few heavy
atoms)
 MAD
Anomalous
scatterering
atoms (Se, Hg,
Br)
guess the
phase:
need a good
model (6D
problem for
finding
orientation)

 How do you judge an X-ray structure’s quality?
 Look at statistics table
1) Resolution
In crystallography,
 > 3
...
0 A: a few errors in backbone, approximate side chains, atom positions to 0
...
5 A: good backbone, some side chains errors, atom positions to 0
...
5 A
reliability factoror the Rvalue or RWork) is a

2
...
15-0
...
5: precise atom locations, individual atoms as separate balls
between
the crystallographicmode
l and the experimental Xray diffraction data
...


2) R-factor =

∑ |Fo −F c|
∑ Fo

, agreement between observed and calculated Fs (spot

intensities/amplitudies)
 when model matches the obs data, R-factor = 0
 anything about 50 % is weird => model doesn’t
match the data
 50 % is random, but 49 % is NOT
 anything above 30% is bad, 25% marginal, 22%
acceptable, anything below 20% is good
 small R-factor = high resolution
*** Discussion about linear fits, parabola, and models/prediction
Free R-factor
A residual function calculated during structure refinement in the same way as the
conventional R factor, but applied to a small subset of reflections that are not used in the
refinement of the structural model
...
5 °


Bonds < 0
...
The sample is placed
in a magnetic field and the NMR signal is produced by excitation of the nuclei sample with radio
waves into nuclear magnetic resonance, which is detected with sensitive radio receivers
...


Certain isotopes have an intrinsic atomic momentum
 With no magnetic field, these are randomly oriented
 With magnetic field, they tend to align with or
against the field according to their nuclear spin

1

quantum number( ∓ 2 )


Now there are two populations of different
spins, with different energies depending on
their alignment



Precession is a wobbling motion that occurs when a spinning object is the subject of an
external force
...


 Chemical shift is the resonant frequency of a nucleus (se of protons) relative to a standard in a
magnetic field
...
e
...
e
...

o This is achieved by producing the
recombinantly in bacteria grown with 15N-ammonium chloride
13
C-glucose as the sole nitrogen and carbon sources respectively

Structures

E
 Basic method for protein structure determination by NMR
1)
2)
3)
4)

Assign all the peaks using COSY-type spectra
Identify all cross peaks assigned diagonal peaks on NOESY spectra
Convert NOESY cross-peaks to distance constrains between corresponding protons
Find 3D structure which optimally satisfies distance constraints as well as protein stereochemistry

 NMR Structure Determination



Molecular modeling with Energy Function

Etotal =E covalent geometry + E NOE restraints
You will get a lot of structures, but use optimization
algorithms to find the molecular structure with the lowest
value of Etotal, which still satisfies all NMR-derived
distance constraints
Generate family of structures
generally not as good as X-ray, but uncertainty may be better



 Resolution
reflection of molecules in-vivo

 Uses of NMR






structures
pka measurements (side chains)
ligand binding
folding
dynamics
o line-shape analysis
o H-D exchanges



---