(Proteins and Enzymes) Diffraction Methods

name 4 diffraction methods

- crystallographic methods

- solution scattering

- fibre diffraction

- electron microscopy

SAXS produces a radial distribution curve from a solution of a purified protein - how can you determine protein structure from this ?

software gives a low resolution structure which a high resolution structure can fit into

What is the significance of SAXS analysing proteins in solution ?

- no constraint of the protein molecule

- can determine protein is flexible

X-ray crystallography uses a 3D crystal lattice structure to predict structure - what kind of structure is produced ?

the highest resolution model where individual amino acids and atoms can be seen

what is an IDD ?

intrinsically disordered domain - proteins may never fold to have a structure

what are the main stages to protein crystallography ?

- crystallisation

- diffraction

- phasing

- model building

what 2 features of a solution would you change in order to change the solubility of the protein in solution ?

pH and salt concentration

describe the protein when pH is at the proteins isoelectric point

net charge is 0 so protein is least soluble

describe the process of salting in/out

- initially add salt to make protein more soluble (affects side chains)

- point where salt removes water form solution making protein less soluble

how does vapour diffusion create a concentrated protein solution which then promotes crystal growth?

water is driven out of protein solution by thermodynamics

how are membrane proteins extracted ?

- detergents

- SMALPS

what is the disadvantage of cleaving proteins with proteases and then crystallising fragments ?

less precise

what is the advantage of using recombinant DNA technology to design and produce specific protein fragments for crystallisation ?

- more precise

- engineered protein can be over expressed in host

what 4 techniques are used to predict protein structure and design a protein ?

- alpha fold

- hydropathy

- IDD predictor

- secondary structure predictor

what is hydropathy?

measure of hydrophobicity of residues - high hydrophobic AA means trans membrane proteins

Q: What rotational symmetries are allowed in crystals, and why?

Crystals only allow 1‑, 2‑, 3‑, 4‑ and 6‑fold rotational symmetries because these are the only rotations that can tile 3D space periodically without leaving gaps.

1‑fold (360°) — identity rotation

2‑fold (180°) — common in orthorhombic/monoclinic crystals

3‑fold (120°) — trigonal/hexagonal systems

4‑fold (90°) — tetragonal systems

6‑fold (60°) — hexagonal systems

Forbidden:

5‑fold (pentagons cannot tile space)

7‑fold, 8‑fold, etc. (angles do not divide 360° evenly)

How does hydropathy affect protein folding?

hydrophobic residues avoid water and hydrophilic residues interact with it.

combination symmetries

screw symmetry

mirror symmetry

glide symmetry

inversions symmetry

What is the effect of using a His-tag to purify a protein in Ni chromatography ?

- His binds to Ni on column

- desires protein is retained in the column while others are washed out

- TEV protease cleavable linker then 2nd affinity column to remove excess tag and tagged TEV protease

in His-tagging Ni chromatography how is the protein removed form the column ?

using a His-tagged protease which cleaves the His-tag and releases pure protein

what is a unit cell of a crystal lattice ?

the basic building block of the crystal

translatied infinitely in three dimensions

what is the asymmetric unit ?

the smallest unit of the unique part of the crystal - accounts for crystallographic symmetry

may contain more than one protein molecule which are squence and chemically identical

but not structurally

when would non-crystallographic symmetry occur ?

asymmetric unit contains multiple protein molecules that are sequence and chemically identical but not structurally identical

what does the diffraction pattern give ?

asymmetric unit

Why do you not see 5-fold, 7-fold, or 8-fold symmetries ?

they are not compatible with the tessellation in lattice

cannot tile 3d space periodically

create a repeating lattice with gaps and overla[s

why do you not see mirror and inversion symmetries in crystals of biological material ?

- proteins are chiral

- to have mirror/inversion symmetry AA would have to be achiral or proteins have an equal no. L and D AA

- does not occur biologically

if 7fold symmetry is not possible how does 7 fold rotation structure of heat shock GroEL crystallise

crystal lattice uses only allowed symmetries - the GroEL particle itself has 7 fold symmetry but the packing arrangement in the crystal does not

according to Braggs law - what has to happen for reflected waves to produce a diffraction spot?

have to be in phase overlap of waves

the extra distance travelled by the lower wave is 2d.sin0

for reflected waves to be in phase the extra distance must be exactly a integral number of wavelengths

protein lattices are just like lots of mirrors - x-ray waves are reflected off of each layer - what is the distance between reflected waves?

the distance that they have travelled

what is the point of the rotation in x-ray diffraction ?

to get the right orientation of the lattice so you get in phase overlap of the wavelengths

what is the real space ?

crystal space

what is the reciprocal space ?

diffraction pattern

how can you get from the reciprocal space to the real space mathematically

fourier transformation

diffraction pattern is capturing most of the FT of the lattice structure in Real space

the reciprocal space data is possible to determine the structure of the real space lattice and its contents

what is diffraction data analysis

the process of turning raw diffraction images (spots) into usable numerical data (indexed, integrated, scaled reflections) that can be used for phasing and structure determination.

looks for systematic absences

symmetry in intensities

lattice constraints

what is indexing in diffraction data analysis ?

identifying the h k and l values of each spot

crystal orientation

lattice type

unit cell dimensions

what is the intensity in diffraction data analysis ?

integration of the x-ray photon counts in each spot

how bright the spot is

how much background noise is present

what is resolution of the diffraction pattern dependent on ?

the degree of order within the sample

mostly limited by the crystal

d value

what is the resolution of higher scattering angles ?

high resolution more data points contribute to the FT structural calculations resulting in a more detailed model

cf visbible microscopy

the process of distinguishing individual parts of an object when examining it with radiation

what does a more ordered crystal structure mean for resolution ?

better packing of molecules so a higher resolution structure

at 8A , 1A, 2A, 4A what can you see

8 = moleuclar shape/envelope

1 = hydrogens being resolved

2 = side chains and cofactors well resolved with few errors, water molecules visible and modelled

4 = tertiary fold and secondary structure some side changes incorrect rotamers

describe the fourier transformation

breaking down repeating pattern in real space to get repeating patterns of waves in the reciprocal space

convert a periodic function to an inverse periodic function via an infinite series of sines or cosines

what doing a fourier transformation what defines the shape of the combines wave more - the waves at high frequency or at low frequency ?

waves at low frequency - high frequency give finer details

high resolution - lower values of d, higher values of h, k, and l higher orders of diffraction

lower resolution - lower orders of diffraction, larger values of d

what is the phase property of a wave?

angle varying between 0-360º and is the amount of delay of one wave wrt to another

what are the wave parameters for FT

amplitude = how strong the wave is - spot intensity - high amplitude = strong contribution

phase = where the wave starts - its horizontal shift = which diffraction does not give you (phase problem) - where the atoms appear - wrong phases = scrambled map

frequency = how many oscillations per unit distance - corresponds to resolution - high frequency = sharpen atomic detail

wavelength = distance between peaks of the wave - small d = high resolution = high angle spots

electron density is a complex repeating pattern of sine waves - what does each peak correspond to ?

atom position and size

phases cannot be experimentally determined - what 2 methods are used to predict them ?

- molecular replacement phasing

- heavy atom phasing

describe the process of molecular replacement phasing

- FT to rotate and translate known section of protein until it matches the data of the unknown

- know some and then build more structure

molecular replacement phasing relies on a part of the protein structure being known - where does this come from ?

- crystallographic models of same protein

- alpha fold predictions

- NMR models

describe heavy atom pushing

- crystals soaked in heavy atoms

- diffraction of heavy atom lattice - native crystal

- pattern of heavy atoms helps determine structure

how would you take a SAXS experiment of a complex made of 2 proteins ?

size exclusion column

what is produced from neutron crystallography?

nuclear density maps

describe structure based ligand design

new drugs are designed to fit the structure of the ligand binding pocket

fragment assembly methods use high throughput crystallography to identify drug fragments that bind

successful fragments are linked together to form high affinity ligands

successful at picking out ligands other methods cant see eg HTS

what is time resolved crystallography

trigger reaction in a cell eg by light substrate diffusion or temperature

collect diffraction at different time delays

compute electron density for each time point

using fourier transforms

build structures for each time point

can see intermediate states, conformational changes etc

XFEL = pulses hit crystals at different time points

ligand discovery example

unexpected electron desnity sandwiched between proteins discovery of inositol phosphate as a key regulator of histone deactylases

same molecule and mechanism regulating species from yeast to humans

key breakthrough in cancer drug development

whats the difference between hanging and sitting vapour diffusion

sitting

- takes 5 days use streak seeding using a rabbit whisker

- dont use additives

- salt = (NH4)2SO4

- buffer = MES

hanging

- takes 7 days use streak seeding use a cat whisker

- precipitant = PEG

- buffer - imidiazole

- additives MnCl2 MgCl2 KH2PO4

what does PEG do

increases viscocity grabs water molecules and improves crystallisation