List of Projects for TSMB '16-'17

Note: Make sure you read the paragraph describing each project carefully before making your choice. Students who produce their own clear, relevant diagrams, using appropriate software, will be marked significantly higher than those who copy equally relevant diagrams from elsewhere with due acknowledgement.

Structural Studies of Small Heat Shock Proteins

Small heat shock proteins are involved in the stabilisation of partially folded proteins. Although the proteins themselves are quite small, they form larger oligomeric complexes. This means that they have been studied by solution state and solid state NMR, X-ray crystallography and electron microscopy. Discuss what each of these techniques has shown for these proteins and what structural biology has contributed to our understanding of these proteins.

NMR of larger proteins and complexes

NMR has been traditionally limited to fairly low molecular weights. Improvements in technology over the last decade or so have allowed the study of larger oligomers (>40kDa) by NMR. Discuss these techniques and the biology they have helped us to understand.

Membrane protein crystallisation

Membrane proteins are hard to express and hard to crystallise once they have been expressed. There are a number of methods of crystallising membrane protein. The project should discuss the methods used and whether they are specific to membrane proteins or are useful for all proteins, but have been used for membrane proteins because the importance of the membrane protein structure justifies the extra labour.

Structural Studies of autophagy

The 2016 Nobel Prize for medicine went to Prof Ohsumi for his discovery of autophagy. The Structure of the proteins of the autophagy system have been studied for the last 10-15 years. This project will summarise the extent of studies of single proteins and complexes involved in autophagy, what they have told us about the mechansim and outline what is still to be discovered.

Structural Biology of the kinetochore

The Kinetochore is the crucial organisational assmebly in cell division and understanding the stucture of the assembly and its components is a topic of current interest in structural biology. The project will review current knowledge of the structure of the kintetochores and its components and discuss the methods used and how the complement each other.

Increasing the resolution limit for Protein Electron Microscopy

The project should survey the improvements in the resolution of electron microscopy structures, the method developments that have led to them, and what can be gained from the greater resolution. Although a survey based on the PDB or EMDB would be good to include in this project, it should be remembered that deposition of EM maps and fitted structures is not compulsory and such a survey will be partial.

Advances in cloning and ligation technology

Classical molecular biology involved the use of restriction enzymes to cut and DNA ligases to join pieces of DNA. As partially covered in TSMB section 2 there are now several newer methods of joining DNA to make expression plasmids etc. The project will critically discuss these advances and their application particularly in the area of high throughput structural biology.

Is it really a water?

There is a tendency to assign most blobs of density in the solvent regions of a protein crystal to water. However other ions such as Na⁺ and Cl^- will appear to be very similar. There is a small literature around these misattributions and possibly stereochemical and experimental means of resolving these ambiguities. As an entry into this topic see a bulletin board discussion https://www.jiscmail.ac.uk/cgi-bin/webadmin?A1=ind1106&L=CCP4BB#34. A good project will involve the student finding a few unattributed non-water atoms in a PDB file (pick one with a resolution in the range 1.8-2.1 Ångstroms).

Femtosecond nanocrystallography

A seminal paper http://www.nature.com/nature/journal/v470/n7332/full/nature09750.html reported the determination of a crystal structure using a femtosecond pulsed X-ray laser. The project will describe in some detail how this new method works and its potential. Review various discussions on the CCP4 bulletin board (google "ccp4bb X-ray laser"). THIS PROJECT PROBABLY REQUIRES A PHYSICS DEGREE AS BACKGROUND.

Low resolution X-ray Crystallography

There are about 100 X-ray crystal structures published at a resolution of 4 Ångstroms or worse since January 2015. The advanced search facility at the RCSB web site should rapidly produce a list. This project will take a selection of these structures and report on what the papers conclude commenting critically on whether the conclusions are justified at the resolution of the structure and on the methods used to derive the model the structure is reporting.

Protein-Protein Interactions Databases

There are now many databases that hold data on protein-protein interactions, both those that have been determined experimentally and those that have been predicted using bioinformatics, but the reliability of these have been questioned. This project involves comparing the techniques used to obtain the interaction data held in these databases and the range of the data available in each of these databases. How reliably are interactions that have been verified experimentally being predicted?

Protein Solubility

Most of the protein structures in the PDB have been determined from aqueous solutions. Discuss the factors that determine protein solubility. If a recombinant protein expressed in E. coli goes into an inclusion body, what strategies can be used to gain a 3D structure? Give examples of structures that have been determined from proteins purified from inclusion bodies and the methods used as well as examples of methods used to alter the expression of proteins so that they are soluble.

Attenuated total reflection (ATR) infra-red spectroscopy of proteins

The high sensitivity of modern ATR Fourier transform infra-red spectrometers and the recent development of time-dependent and equilibrium methods to obtain accurate difference spectra (notably by the groups of Klaus Gerwert and Peter Rich) are able to reveal details of changes in structure, hydration and protonation states in protein complexes as a result of changes in pH, redox potential or ligand binding. This project will outline the principles underlying these methods and survey their application and potential to advance our mechanistic understanding of protein function.

Electron Microscopy, Crystallography and Molecular Modelling

Detailed molecular structures of many large protein complexes or "molecular machines" have been produced using a combination of techniques. This is done by fitting atomic resolution structures of individual protein chains (or even domains) into the lower-resolution electron density maps from electron microscopy (or from low resolution X-ray crystallography). Describe the methods for fitting structures determined by X-ray crystallography or NMR into low resolution maps and give examples of how this has been used in practice, what biological insight it has given, and the limitations that arise from the low resolution.

Methods for Automated Crystallographic Model Building

Increasingly automated methods can be used for building a nearly complete atomic model (PDB) file from an initial experimentally phased electron density map or molecular replacement solution. These programs include ARP/wARP, Bucanner (CCP4), Resolve and Textal (Phenix). The project will outline how the various automated building programs work and describe what applications the methods are effective for and their limitiations.

Measuring progress in protein structure prediction: the CASP series of structure prediction competitions.

CASP (Critical Assessment of Structure Prediction) http://predictioncenter.org/ is a series of biannual "competitions" to assess the quality of protein structure prediction methods. The results of each competition are published in a supplement to the journal Proteins and there are reviews published after each CASP conference. This project will involve reviewing the progress made in structure prediction during the first ten competitions (from 1994 to 2012), and critically discussing the most successful approaches and methodologies.

Surface layer measurement of Affinity

As well as Surface Plasmon Resonance covered in section 11, a number of other techniques that use surface layer binding to measure protein-protein interactions are now on the market. These includ Blitz and Octet from Fortebio which use optical biosensors, and also various piezo-electric methods for measuring protein binding to surfaces. The project will review the methods and give some examples of applications and published results of the methods.

SAXS/SANS (Small angle X-ray and neutron scattering)

Small angle X-ray and neutron scattering can be used to probe the low resolution shape, conformation and oligomeric state of macromolecules. In some cases to a similar resolution as electron microscopy. The project will outline the theory of the methods and concentrate on on at least four detailed examples where the results have given biological insight.

FRET (Fluorescence Energy Resonance Transfer) and FLAP (Fluorescence Localisation after Photo Bleaching) in vivo

Fluorescence techniques such as FRET and FLAP are increasingly being used to study protein interaction and mobility in intact cells. Describe these methods, giving examples of the type of studies that can be carried out with the techniques and their limitations.

Please refer to the guidelines before choosing your project.

Nicholas Keep and Clare Sansom, June 2017