Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

proteins > protein X ray Crystallography pt II > Flashcards

protein X ray Crystallography pt II Flashcards

1
Q

method for growing crystals

A

vapour diffusion

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

describe vapour diffusion

A
  • water in drop evaporates into resivoir
    -volume of drop slowly decreases
    -protein concentation slowly increases
    -over time 1 day 4 weeks crystals grow
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what do you need once you have your crustal

A

x ray diffractometer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what are the three components of a x ray diffractometer

A

x ray source
crystal holder
diffraction detector

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

x ray source

A

x ray tube (for home sources typically uses copper)
particle accelerator (synchotron, e.g. CLS)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

crystal holder

A

goniostat/ goniometer (to turn the crystal so you see all the sides of your protein)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

diffraction detector

A

imaging plate
CCD- charge coupling device- camera

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

what are the two consequences of x ray diffraction being a 3D phenomenon

A
  1. a reflection can be described by its coordinate in this 3D space (h,k,l)
  2. have to move around the detector to record as many data points (reflections) as possible= for practical reasons we have to move the crystal
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

what are the auxillary components for x ray crystallography data collection

A

cryogenic (liquid N2) stream
beam stop
camera

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

cryogenic stream

A

to prevent radiation damage of the crystal sample
photon energy can kick off electrons- covalent bonds break

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

beam stop

A

to prevent detector damage
most x rays pass directly through the crystal

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

camera

A

not the CCD camera detector
to centre sample crystal in the path of x ray beam

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

what is the process for harvesing crystals

A

scoop the crystal out with a microscopic loop
freeze sample to prevent drying and radiation damage

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what are the typical dimensions of a protein crystal

A

<1mm in length

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

how is the data processed for x ray crystallography

A
  1. indexing
  2. scaling
  3. merging
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

indexing

A

200-2000 images

reflections are indexed: intensity and h,k,l

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

scaling

A

signal strength is affected by many factors
e.g. how protein molecules are packed in the crystal
- intensity values are normalized

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

merging

A

redundant data points (there should be many) are combined
- all data points (reflections) are tabulated into a single file

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

diffraction pattern

A

list of reflection intensity and coordinate (F(H,K,L))

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

electron dnesity map

A

reconstructed electron cloud (p(x,y,z))

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

plugging the reflection data (structure factor) into the equation, what can we calculate

A

the propability function
p(x,y,z)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

how do we visualize the propability function

A

produce a 4D controur map

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

what is a 4D contour map

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

to fully describe each reflection mathematically, we need to

A

be able to measure both the amplitude and the phase of the X-ray wave producing the reflection

25
phase problem
However, there is no detector that can measure phase so that we can describe the reflection mathematically
26
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
27
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
28
for her determination of x ray techniques of the structures of important biochemical substances penicillin vitamin B12 insulin
dorothy crowfoot hodgkin
29
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:
30
manual refinement
electron density - atomic model
31
automated refinement
diffraction data and atomic model
32
what makes a good structure model
resolution crystallographic R factor agreeement with known values of protein atomic bond lengths and angles
33
resolution
determined by how well the crystal diffracts X rays (degree of order and size) >2A good >1.5 A very good
34
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
35
agreement with known values of protien atomic bond legnths and angles
ramachandran plot want all psi/phi angles to be in the allowed region
36
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
37
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.
38
Cryo-EM
cryogenic electron microscopy a type of TEM
39
what is required to see things in Cryo-EM
object light lens recording/ display device: electron detector
40
object in cryo-em
object- protein molecules- thinly frozen solution sample -prep much easier than x ray crystallography
41
light
2. 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
42
lens
3. 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
43
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)
44
progess of cryo em
made revolutionary progess in recent years- ave res now is 5A hardware: electron detector software: image processing
45
For developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution” Richard Henderson
Jacques Dubochet Joachim Frank
46
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
47
advantages of x ray crystallography
- Well developed - High resolution - Broad MW range
48
disatvantages of x ray
- Difficult for crystallization - Static crystalline state structure
49
objects in x ray
- Crystallizable samples - Soluble proteins, membrane proteins, ribosomes, DNA/RNA and protein complexes
50
resolution of x ray
high
51
NMR advantages
- High resolution - 3D structure in solution - Good for dynamic stud
52
NMR disatvantages
- Need for high sample purity - Difficult for computational simulat
53
NMR objects
- MWs below 40-50 kDa - Water soluble sample
54
NMR resolution
high
55
cryo EM advantages
- Easy sample preparation - Structure in native state - Small sample amoun
56
cryo EM disadvantages
- Relatively low resolution - Applicable to samples of high MW only - Costly EM equipmen
57
cryo EM objects
- MW > 150 kDa - Virions, membrane proteins, large proteins, ribosomes, complex compounds
58
resolution of cryo EM
relatievly low <3.5 A
59

proteins (2 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions