Lectures 6-10: Protein structure and determination

Question 1

where do the structures come from

Answer 1

x ray crystallography - determines atomic positions form crystalline sample

NMR spectroscopy - determines distances between defined proteins from protein solutions and computes models fitting these restraints

electron microscopy - determines atomic positions from vitrified solutions

Question 2

why x rays

Answer 2

microscopy is limited by the diffraction limit

visible light is measured in nanometres

atoms are seperated by distances of the order of 0.1 nm
x rays 10^-10

Question 3

sources of x rays

Answer 3

lab vs Synchrotron
bombarding copper targets with high energy electrons and x ray beams are emitted
not bright
poor quality
not useful for tiny crystals or collecting data quickly

high res = synchrotron
between Winchester and oxford
Diamond light facility
ring of magnets
accelerate in a circular orbit
high intensity
 1 week vs 1 minute

Question 4

why not build an x ray microscope to study protein structure

Answer 4

diffracted rays cannot be recombined to form an image
no lens
a digital computer is used to restructure the information

Question 5

x rays interaction w molecules

Braggs Law

Answer 5

single molecules v weak
impossible to detect above the noise level of a layer of water
crystals are used

crystal - many copies of he molecules on a 3d grid - when x rays strike crystal info detected on reciprocal lattice grid - diffracted waves are in phase w each other = intense black spots in a constructive interference process
any atom that isnt on the planes scatters and isnt in phase detracts from the intensity Braggs’s Law
n lambda is 2 D sin theta
relates distance between the atoms to the angle ifraction
tells us if we have a wave front striking a crystal the scattered wave has to give a diffracted wave which is an integral number of wavelengths in order for the 2 waves to constructively interfere

Question 6

diffraction patterns

Answer 6

as a result of constructive patterns

waves annihilate each other when they are in phase at 180 degrees
spots - reflection these dots measured in x ray exp
measuring geometry and intensity of scatterred waves

Question 7

protein crystallography

Answer 7

formed from supersaturated solutions with respect to the solute
more dissolved substance than is stable in thermodynamic terms
super saturated sol - diff agents to reduce protein conc i.e. ammonium solfate

hanging drop vapour diffusion
resovoir 50% sat with strong ammonium sulphate
drop with protein sol in 25% saturates ammonium sulfate
seal chamber with vacum grease
leave to stand
water vapour extracted from drop
and moves into resovoir
drop shrinks
protein conc goes up
ammonium sulfate conc goes up and your crystals grow in the tiny drop of liquid

sitting drop

Question 8

automation of protein crystallography

Answer 8

each protein requires a diff set of conditions
ph buffers salt precipitants
use of robot
nono crystallisation 
fast hanging or sitting drop experiment
weeks
mosquito robot in portsmouth - positive displacement pipette robot - pipette nano litre volumes 
set up 100 exp in 10 mins

Question 9

the phase problem

Answer 9

diffraction pattern tells up the amplitude of waves
but not their phase
not the relative time of arrival of the waves in constructing the image
phase shift
must regain phase info for each reflection in data set to create 3d structures
phases + diffraction images = electron density

Question 10

solving the phase problem

Answer 10

1. making recombinant protein = use biosynthetic incorp of selenium atoms into amino acid side chain of thymine
   comparing diff patterns of selenium version of protein with native = calculation of phase information
   synchrotron precise measurements of scattering of selenium

determined phases
= electron density map
build polypeptide sequence in extended chicken model

Question 11

crystals vary in precision

Answer 11

this is called resolution
measure in A
the lower the res is the higher the precision of the structure

resolution is determined by how far out from the centre the diffraction spots are
determined by the quality of the crystal

most precise method for determining protein structures

no molecular weight limilit like in NMR spec

rate limiting step is often growth of diffraction quality protein crystals

crystals muct be ismophous with molecules perfectly aligned
iso same
morphous shape

may be inaccuracies in.structures as the molecules are not in solution

Question 12

NMR

nuclear magnetic resonance spectroscopy

Answer 12

each peak arises from one hydrogen nuclei (protons) within the protein

the frequency of each peak (measured in parts per million) is determined by the chemistry and electronic environment of the proton nucleus

Question 13

how is nmr used for protein structure

Answer 13

we identify pairs of hydrogen nuclei that are close together in the folded protein and use this to calculate confirmation of the protein
determine distances
x ray determines atomic positions

Question 14

Resonance assignment of chemical shift assignment

Answer 14

which proton peeks or resonances in the spectrum correspond to which hydrogen atoms in protein

assign each peak to a proton

correlating protons that are connected by up to 3 covalent bonds

by using 2D nmr pulse sequence with radio frequency energy
look at how its dissipated through bonds
can pick out neighbours
2D cosy spectrum

Question 15

indirect structure determination

Answer 15

unlike x ray crystallography we don’t get an electron density map and we can’t see the protein directly

we must calculate models based on experimental geometric restraints

nucelear overhauser effect - identifies pairs of protons that are close together in space

Given enough of these any any restraints, we can calculate a family of structures, an ensemble that is consistent with the measured experimental data.

Question 16

the Noe nuclear overhauser effect ambiguity problem

Answer 16

if either proton chemical shift is non-unique in these interaction maps, then the energy is said to be ambiguous.

To assign ambiguous anyways, we need to know the structure of the protein, but determine the structure We must have signed the NPT

rate limiting step

Question 17

problem solved by iterative method

Answer 17

preliminary protein structures are calculated from the unambiguous NOE’s only
= approximate structures
multiple cycles of reassignment + recalibration
calculated using restrained moleculare dynamic simulation
The restraints are the energy into proton distances and are applied to like rubber bands in the molecular dynamics simulation.
protein sturcuture deposited in databank

Question 18

xray vs nmr

Answer 18

sample highly pure in both ismorphous protein crystal n x ray but a sol in nmr

nmr is demanding of amounts of protein available
because you need something in the region of a one in millimoles solution of your protein.

x ray diffractmeter

diffraction pattern data
vc interproton distances

electron density single structure result vs structure ensemble

precision higher in x ray

the accuracy of the methodology is moderate to high with X-rays, but very high with nmr because working with sol

so limit of size of proteins that can look at in x ray where as structure is in solution in nmr

disadvantage x ray needs to grow size limit in nmr

Question 19

precision vs accuracy

Answer 19

precise by inaccurate - x ray high definition but in semi solid state

accurate but imprecise - nmr - in solution so close to actual structure but rathe hazy

Question 20

the protein databank

Answer 20

had java software for protein strucutre visualization

atomic co ordinates can be downloaded for analysis using other programs

Question 21

Cryo electron microscopy

EM

Answer 21

low dose enables large complex structures that arents available in high quantities to be crystallised

Question 22

light vs electron microscope

Answer 22

the accuracy of the methodology is moderate to high with X-rays, but very high with any more focused with an objective lens. Two producers magnified image of the object, which is then projected through an eyepiece

see cells with nuceli

i micron size limit

transmission electrom microscope
instead of a lamp, we have a source of electrons.

She’s usually a hot tungsten more and we have lenses.

But instead of being glass lenses,
these are electromagnets because electrons are charged particles which have been accelerated through high voltage in this gun.

As they pass through the electromagnetic lenses, they get focussed in a similar way to light in a condenser lens.

Then they hit a specimen and then there is an objective lens with an electromagnet and a projector lens.
Another electromagnet, which typically projects the image onto a fluorescent screen.

the magnified image can be seen at the bottom of the microscope

the differences between the microscope and the transmission electron microscope use it
instead of visible light like the ESM uses a stream of electrons exaggerated to high speed,
and the wavelength of these accelerated electrons is very, very short.
must be kept under vacuum otherwise air will scatter the electrons

electron:
In order to get samples into the microscope, we have to use an air lock system where you shut the airlock. Bring a sample carrier. Load your samples onto the carrier and put them back in and then open the airlock.

visible microscope resolutions limits to half the wavelength
= 3000 units

limit caused by lens quality
electromagnetic lenses are no perfect
and sample stability

Question 23

negative staining with an electron microscope

Answer 23

Electron microscopy of biological specimens involved fixing the samples and cutting thin sections and then staining them with a heavy metal salt. embedding the sample in a resin i,e, uranium acetate
when the beam is turned on in the electron microscope, the actual sample turns to ashes because there’s so much energy in the beam. all that remains is the heavy metal cast

Question 24

to determine atomic structures

Answer 24

henderson and Unwin 1975

the first determination of a protein structure by EM the first image of a membrane protein

on each molecule of this proton pump, there is a covalently attached molecule of retinal over attach to a lysine

involves in sensing of light
change in structure of protein pump in response to light
= proton translocation across membrane
bacteria redopsin
retinol attaches to lysine residue
the chemical reaction takes place when photon light strikes there’s proton pump means that it changes.
The bonding pattern in the retinal form are all crowns, double bond type of structure.
This has got alternating double bonds all the way through or On absorption of light. This chain gets kinked.

combining tilted images = 3D image of an object
6A resolution = tubes

lpc method electron crystallography
diffraction apttern

Question 25

modern phase

Answer 25

cropreservation
protect from radiatio damage take it down to low temperatures
rapid immersion of samples in liquid ethane cools them so fast that there is no time for ice crystal formation
the same is trapped in vitreous ice
turns into glass

developments for single particle
1. field emmission electron gun
2. improves magnets
3. phase plaes
4. low tempt
5. rapid read out electron detectors for recording movies as they vibrate a little
now we have
1. highly urifies samples
2. single particles trapped in vitrified water at v low temps in iff orientations
3. check out particles by negative stain
4. record thousands of images of vitrifiied sample
5. perform corrections for movement, spherical aberration, astigmatism , defocus contrast
6. put the images in classes of orientation
7. Average to improve signal to noise
8. combine projections for 3D image
9. Fit pre existing X ray structures or trace the polypeptide chain in the density map that can be produces from this imagine in the electron microscope

Question 26

can we predict a structure straight from an amino acid sequence

Answer 26

GLu met ALaGlu Lys Ala Lys = a helix
tyr iLe Val Phe Trp Ile Tye = beta strand
asn tro Gly pro Asp Gly Pro = turn region

algorithums for preditcting secondary structures
1. Chou-Fasman

for a helix: scan for a window of 6 residues with a score greater than 4

beta strands:

2. GOR Garnier-Osguthorpe-Robson
3. Neural Networks

b strands can be identified but they cant be linked to beta sheets

accuracy 40-60%

Question 27

Ab. Initio prediction of secondary structure vs homology modelling

Answer 27

prediction program
secondary structure content
try fold into tertiary

homology modelling
- Put your sequence through database searches and
8:41
find a protein with a similar sequence where the structure is actually known.
And then you can use the name structure as a template.
if we have a crystal structure of a homologous protein we can model the structure of the protein we are interested in

Question 28

multiple sequence alignment

Answer 28

identify structural and functional important residues through their invariance or high conservation
alignments r one by eye or comp

SWISSMODEL

Question 29

mutation data matrix

Answer 29

applies a score to the alignment of each residue pairing for conservation

gap penalty = -3

BLAST

Question 30

homology model protocol

Answer 30

1. start with known atomic co ordinates
2. add/remove gaps in backbone
3. delete side chains of mutant residues
4. rebuild new residue side chains
5. energy minimise whole structure