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proteins > protein X ray Crystallography pt II > Flashcards

protein X ray Crystallography pt II Flashcards

1
Q

method for growing crystals

A

vapour diffusion

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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
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3
Q

what do you need once you have your crustal

A

x ray diffractometer

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4
Q

what are the three components of a x ray diffractometer

A

x ray source
crystal holder
diffraction detector

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5
Q

x ray source

A

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

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6
Q

crystal holder

A

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

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7
Q

diffraction detector

A

imaging plate
CCD- charge coupling device- camera

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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
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9
Q

what are the auxillary components for x ray crystallography data collection

A

cryogenic (liquid N2) stream
beam stop
camera

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10
Q

cryogenic stream

A

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

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11
Q

beam stop

A

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

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12
Q

camera

A

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

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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

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14
Q

what are the typical dimensions of a protein crystal

A

<1mm in length

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15
Q

how is the data processed for x ray crystallography

A
  1. indexing
  2. scaling
  3. merging
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16
Q

indexing

A

200-2000 images

reflections are indexed: intensity and h,k,l

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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

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18
Q

merging

A

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

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19
Q

diffraction pattern

A

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

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20
Q

electron dnesity map

A

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

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21
Q

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

A

the propability function
p(x,y,z)

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22
Q

how do we visualize the propability function

A

produce a 4D controur map

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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

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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
Q

phase problem

A

However, there is no detector that can measure phase
so that we can describe the reflection mathematically

26
Q

solution to the phase problem

A

Most commonly used solution is “molecular replacement” `where the phase information is deduced from a previously determined structure of a similar protein

27
Q

personal bias in electron density interpretaiton of x ray crystalography

A

must decide from the high resulution data where to put each atom in the protein and which amino acid fits whre

28
Q

for her determination of x ray techniques of the structures of important biochemical substances
penicillin
vitamin B12
insulin

A

dorothy crowfoot hodgkin

29
Q

model building and refinement of x ray crystallography

A
  • 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
Q

manual refinement

A

electron density - atomic model

31
Q

automated refinement

A

diffraction data and atomic model

32
Q

what makes a good structure model

A

resolution
crystallographic R factor
agreeement with known values of protein atomic bond lengths and angles

33
Q

resolution

A

determined by how well the crystal diffracts X rays (degree of order and size)

> 2A good
1.5 A very good

34
Q

crystallographic R factor

A

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
Q

agreement with known values of protien atomic bond legnths and angles

A

ramachandran plot

want all psi/phi angles to be in the allowed region

36
Q

characterization of drug target interactions by X ray crystallographt is standard in modern drug dyscovery efforts

A

E.g., Nirmatrelvir
- SARS-CoV-2 Mpro inhibitor
- Active ingredient in Paxlovid

37
Q

Protein X ray crystallography limitations

A
  • 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
Q

Cryo-EM

A

cryogenic electron microscopy

a type of TEM

39
Q

what is required to see things in Cryo-EM

A

object
light
lens
recording/ display device: electron detector

40
Q

object in cryo-em

A

object- protein molecules- thinly frozen solution sample
-prep much easier than x ray crystallography

41
Q

light

A
  1. 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
Q

lens

A
  1. 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
Q

recording/ display device

A

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
Q

progess of cryo em

A

made revolutionary progess in recent years- ave res now is 5A

hardware: electron detector
software: image processing

45
Q

For developing cryo-electron microscopy for the high-resolution structure
determination of biomolecules in solution”
Richard Henderson

A

Jacques Dubochet Joachim Frank

46
Q

disatvantages of cryo EM

A
  • 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
Q

advantages of x ray crystallography

A
  • Well developed
  • High resolution
  • Broad MW range
48
Q

disatvantages of x ray

A
  • Difficult for
    crystallization
  • Static crystalline state
    structure
49
Q

objects in x ray

A
  • Crystallizable samples
  • Soluble proteins,
    membrane proteins,
    ribosomes, DNA/RNA
    and protein complexes
50
Q

resolution of x ray

A

high

51
Q

NMR advantages

A
  • High resolution
  • 3D structure in
    solution
  • Good for dynamic
    stud
52
Q

NMR disatvantages

A
  • Need for high sample
    purity
  • Difficult for
    computational
    simulat
53
Q

NMR objects

A
  • MWs below 40-50 kDa
  • Water soluble sample
54
Q

NMR resolution

A

high

55
Q

cryo EM advantages

A
  • Easy sample
    preparation
  • Structure in native
    state
  • Small sample
    amoun
56
Q

cryo EM disadvantages

A
  • Relatively low
    resolution
  • Applicable to samples
    of high MW only
  • Costly EM equipmen
57
Q

cryo EM objects

A
  • MW > 150 kDa
  • Virions, membrane
    proteins, large proteins,
    ribosomes, complex
    compounds
58
Q

resolution of cryo EM

A

relatievly low
<3.5 A

59
Q
A

proteins (2 decks)

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