X-ray crystallography Flashcards

1
Q

What does the position of a diffraction spot indicate?

A

level of detail/resolution

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2
Q

What does the amplitude of a diffraction spot indicate?

A

the types of atoms

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3
Q

What is the phase problem?

A

diffraction spots miss information about the relative positions of the atoms
-use molecular replacement (using similar, known structure as a model) or isomorphus replacement (add heavy metal atoms and compare diffraction patterns, heavy atoms easy reference points) to solve phase problem

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4
Q

How is the phase problem solved?

A

molecular replacement
-similar, known structure used as a model
-orientations and positions optimised to get good match between calculated and expected diffraction patterns

isomorphus replacement
-add heavy metal atoms to protein
-compare diffraction patterns
-heavy metal atoms act as easy reference points to determine the phase

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5
Q

How are protein crystals for x-ray crystallography obtained?

A

vapour diffusion
-conditions brought to a point where the protein is supersaturated and forms an ordered crystal

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6
Q

What makes membrane proteins difficult to study using x-ray crystallography?

A

-bilayer must be disrupted
-proteins aggregate in water
-detergent required

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7
Q

What are detergents?

A

amphiphilic water soluble surfactants
-polar head group, non-polar hydrophobic chain
-like lipids but more soluble

eg. alkyl maltopyranoside family of detergents popular for membrane protein studies

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8
Q

What is the critical micellular concentration (CMC)?

A

the concentration where the substances (detergent, membrane lipids and proteins) self-associate into micelles
-detergent concentration needs to be above CMC

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9
Q

How can the contact protein crystals have with detergent be increased (for small proteins)?

A

-fusing protein to larger protein domain
-partnering protein with small hydrophilic proteins (eg. Fab or Fc antibody fragments)

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10
Q

Structure of protein crystals

A

repeating lattice
-network of weak intermolecular interactions
-large channels/spaces in lattice -> get filled by water/buffer

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11
Q

How can the scattering pattern be analysed?

A

Using Bragg’s law, in which the reflected waves are interfered by a whole number of wavelengths

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