probio analysis of structures

Traditional and Modern Versions of x-ray sources?

Traditional: bombarding pieces of metal with high energy electrons.

Modern - synchotrons, much brighter, faster data collection, minimizing crystal damage and labor, but there is only 10 in the world.

Synchotrons

are advanced particle accelerators that produce intense beams of light, enabling high-resolution imaging and analysis. They use the motion of charged particles in magnetic fields to generate synchrotron radiation, which is used in various scientific applications.

APS

an example of a synchrotron facility, specifically the Argonne National Laboratory's Advanced Photon Source, which provides X-ray and other forms of radiation for research.

Phase Issue in X-ray cyrstallography

Xray diffraction only detects differences in amplitudes, not phases. Depending on phase, waves may strengthen or cancel each other.

Ways to solve the phase problem

Isomorphous replacement, Molecular Replacement, ROSETTA, MAD, Direct Methods

Isomorphous replacement

Comparison to crystals formed by modified protein (with addition of heavy atoms)

Molecular Replacement

Comparison to related proteins of similar structures, uses ROSETTA to relate to known structures

Multiwavelength anomalous dispersion (MAD)

Measures variation of the intensity distribution in the diffraction pattern over a range of wavelength, uses some atoms to absorb/scatter X-rays

Direct method

Uses knowledge of general features of e- density distrbution in crystals

What are the two approaches to interpret electron density data?

Old (superimposing electron density maps)

new (computer graphics, FRODO → O)

How is the structure completed?

Iterated model rebuilding/recomputation: XPLOR and ARP/wARP

XPLOR

progressive model rebuilding program

ARP/wARP

fitting models automatically

How is the computations refined?

compares the observed structure factor magnitudes to the magnitudes of structures made by the model

What are the resolution numbers?

4 A: General bonding/helices

2.8 A: Mainchain, some side chains

2.3 A: Better resolution of features

1.8 A: Clearly identified Residues

Around 2 A is atomic resolution, but 2.5-3 A is “Near atomic” resolution

Advantages of xray crys?

Most precise for protein structure

can determine entire protein complexes

downsides of xray crys?

proteins with hydrophobic sequences cannot be in water

fibrous proteins

proteins that are intrininsically disordered (will denature anyway)

proteins that may form amyloids will misfold

to fix, crystallize regions rather than full proteins or use other techs

NMR Spec

Uses the spinning of isotopic atoms to find structure: strong magnetic fields to measure transitions of different nuclear spin states

Advantages of NMR?

No crystals needed

can solve large structures

can provide info on protein structure dynamics

Disadvantages of nmr?

requires isotopic labeling

provides a range of possible structures (15-20) other than one

COSY

correlation spectroscopy: values of conformational angles between bound atoms

NOE

nuclear overhauser effect, identify non-bound atoms that are close to each other in structure and find out how far

Residual bipolar couplings

find the orientation of cdertain segments with respect to external magnetic field

TROSY

Transverse relaxation optimized spectroscopy: using sharper signals to analyze large molecules

solution state nmr

nmr in liquid

longer rotational signal

weaker signals are harder to interpret

good for dynamics

solid state nmr

good for determining structures

alternative for structures unable in xray crys

How to pick between the nmr set of possible structures?

stereochemical deviations

some structures arent possible

experimental data

cryo-em

freezing in liquid nitrogen/ethane, no crystal ice

no size limit, less quality (3-4 A)

e- crys

uses electron diffraction from 2-d crystals

image reconstruction

combine multiple images of the same object

how is cryo em improving?

development of thin, sensitive layer to record electrons that does not scatter the signal

contrast to direct electron detectors, used film and charge-coupled devices

software: bayesian stats, account for noise and heterogeneity in a sample

single particle cryo em reconstruction

using a bunch of small images, put together for one structure

FSC

Measuring resolution through fourier shell correlation by splitting data in half and fitting them together

overfitting

iterating and modeling noise

non-uniform resolution

some dynamic proteins will have different structures

symmetry problems

too many repeated structures cause problems because distinctive features may not be recognized

PDB

protein data bank: library of structures

homology modeling

using another structure that has 30-40% similarity to model a new protein

fold recognition

look for motifs and patterns and structures that make favorable interactions

2ndary structure predictions

provides sequences that can form secondary structures

novel fold prediction

minimum energy conformation

dynamic simulation

least detailed model giving correct answer