protein X ray Crystallography pt II Flashcards
method for growing crystals
vapour diffusion
describe vapour diffusion
- water in drop evaporates into resivoir
-volume of drop slowly decreases
-protein concentation slowly increases
-over time 1 day 4 weeks crystals grow
what do you need once you have your crustal
x ray diffractometer
what are the three components of a x ray diffractometer
x ray source
crystal holder
diffraction detector
x ray source
x ray tube (for home sources typically uses copper)
particle accelerator (synchotron, e.g. CLS)
crystal holder
goniostat/ goniometer (to turn the crystal so you see all the sides of your protein)
diffraction detector
imaging plate
CCD- charge coupling device- camera
what are the two consequences of x ray diffraction being a 3D phenomenon
- a reflection can be described by its coordinate in this 3D space (h,k,l)
- have to move around the detector to record as many data points (reflections) as possible= for practical reasons we have to move the crystal
what are the auxillary components for x ray crystallography data collection
cryogenic (liquid N2) stream
beam stop
camera
cryogenic stream
to prevent radiation damage of the crystal sample
photon energy can kick off electrons- covalent bonds break
beam stop
to prevent detector damage
most x rays pass directly through the crystal
camera
not the CCD camera detector
to centre sample crystal in the path of x ray beam
what is the process for harvesing crystals
scoop the crystal out with a microscopic loop
freeze sample to prevent drying and radiation damage
what are the typical dimensions of a protein crystal
<1mm in length
how is the data processed for x ray crystallography
- indexing
- scaling
- merging
indexing
200-2000 images
reflections are indexed: intensity and h,k,l
scaling
signal strength is affected by many factors
e.g. how protein molecules are packed in the crystal
- intensity values are normalized
merging
redundant data points (there should be many) are combined
- all data points (reflections) are tabulated into a single file
diffraction pattern
list of reflection intensity and coordinate (F(H,K,L))
electron dnesity map
reconstructed electron cloud (p(x,y,z))
plugging the reflection data (structure factor) into the equation, what can we calculate
the propability function
p(x,y,z)
how do we visualize the propability function
produce a 4D controur map
what is a 4D contour map
connect all (X,Y,Z) coordinates that give the same probability value p on a 3D plot
gives a 3D shape enveloped at that propability level - electron density map
to fully describe each reflection mathematically, we need to
be able to measure both the amplitude and the phase of the X-ray wave producing the reflection
phase problem
However, there is no detector that can measure phase
so that we can describe the reflection mathematically
solution to the phase problem
Most commonly used solution is “molecular replacement” `where the phase information is deduced from a previously determined structure of a similar protein
personal bias in electron density interpretaiton of x ray crystalography
must decide from the high resulution data where to put each atom in the protein and which amino acid fits whre
for her determination of x ray techniques of the structures of important biochemical substances
penicillin
vitamin B12
insulin
dorothy crowfoot hodgkin
model building and refinement of x ray crystallography
- Building a model into electron density is not a trivial process
(typically hundreds of a.a.s) ⇨ initial models are inaccurate - Improving the accuracy of your model = refinement
- Refinement is an iterative process:
manual refinement
electron density - atomic model
automated refinement
diffraction data and atomic model
what makes a good structure model
resolution
crystallographic R factor
agreeement with known values of protein atomic bond lengths and angles
resolution
determined by how well the crystal diffracts X rays (degree of order and size)
> 2A good
1.5 A very good
crystallographic R factor
measure of agreement between the structrue factors calcuoalted from the model and those from the original diffraction data
typically 0.18-0.25 range
<0.2 good
<0.15 very good
agreement with known values of protien atomic bond legnths and angles
ramachandran plot
want all psi/phi angles to be in the allowed region
characterization of drug target interactions by X ray crystallographt is standard in modern drug dyscovery efforts
E.g., Nirmatrelvir
- SARS-CoV-2 Mpro inhibitor
- Active ingredient in Paxlovid
Protein X ray crystallography limitations
- Protein must be crystallizable
- Often takes long to figure out the right condition
- Some just do not crystallize!
- Resolved structure is static
- Proteins are inherently dynamic entities
- Structure may contain crystallographic artefacts
- Crystallization conditions are typically not physiological
E.g., acidic/basic pH, high concentrations of precipitant, salt,
ligand, etc.
Cryo-EM
cryogenic electron microscopy
a type of TEM
what is required to see things in Cryo-EM
object
light
lens
recording/ display device: electron detector
object in cryo-em
object- protein molecules- thinly frozen solution sample
-prep much easier than x ray crystallography
light
- Light: electrons (also have wave properties)
The “ingredients of seeing” specific for cyro-EM:
Cryogenic electron microscopy (Cryo-EM)
- Electron gun shoots out electrons at the sample
- At high speed (energy), the wavelength approaches 0.1 nm
- Scattered off the electrons in the sample
lens
- Lens: electromagnetic fields
The “ingredients of seeing” specific for cyro-EM:
Cryogenic electron microscopy (Cryo-EM)
- Unlike photons, electrons are charged
- Scattered electron beams can be refocused to create an image
recording/ display device
Recording/displaying device: electron detector
The “ingredients of seeing” specific for cyro-EM:
Cryogenic electron microscopy (Cryo-EM)
- Technology lacked in this regard
⇨ Produced low resolution structures (~15 Å)
progess of cryo em
made revolutionary progess in recent years- ave res now is 5A
hardware: electron detector
software: image processing
For developing cryo-electron microscopy for the high-resolution structure
determination of biomolecules in solution”
Richard Henderson
Jacques Dubochet Joachim Frank
disatvantages of cryo EM
- Resolution is still limited compared to X-ray crystallography
- Average resolution is ~5 Å currently
Disadvantages - Inherent high levels of noise
- More suitable for large complexes (signal-to-noise ratio improves as
the molecular size increases
advantages of x ray crystallography
- Well developed
- High resolution
- Broad MW range
disatvantages of x ray
- Difficult for
crystallization - Static crystalline state
structure
objects in x ray
- Crystallizable samples
- Soluble proteins,
membrane proteins,
ribosomes, DNA/RNA
and protein complexes
resolution of x ray
high
NMR advantages
- High resolution
- 3D structure in
solution - Good for dynamic
stud
NMR disatvantages
- Need for high sample
purity - Difficult for
computational
simulat
NMR objects
- MWs below 40-50 kDa
- Water soluble sample
NMR resolution
high
cryo EM advantages
- Easy sample
preparation - Structure in native
state - Small sample
amoun
cryo EM disadvantages
- Relatively low
resolution - Applicable to samples
of high MW only - Costly EM equipmen
cryo EM objects
- MW > 150 kDa
- Virions, membrane
proteins, large proteins,
ribosomes, complex
compounds
resolution of cryo EM
relatievly low
<3.5 A
