Structural Biology

Protein Crystallography and Structure Determination

X-RAY Crystallography

Protein Crystallography and Structure Determination

Cryo-EM

Protein Crystallography and Structure Determination

Protein NMR

Which structural biology technique should I choose?

Depending on your particular protein target and your project goals, we will help to advise you on which structural biology technique is most appropriate for your project. For a summary of the pros and cons of the three techniques see below.

For an in depth description of the techniques we offer read more in our blog here. 

Advantages Disadvantages
  • Well established technique
  • Can achieve atomic resolution of macromolecular structure in crystalline state
  • High throughput once crystal system established
  • No size limitation of macromolecule
  • Data collection, processing, and analysis is relatively quick
  • Requires sample to crystallise
  • Crystals need to be well ordered to achieve high resolution diffraction
  • Snapshot of conformation
  • Radiation damage – this can be mitigated to some degree
  • Crystal packing can influence conformation
  • Requirement to solve phase of diffraction data to calculate electron density
  • X-ray source (in-house or synchrotron) required with appropriate goniometer, detector, etc needed for data collection
Advantages Disadvantages
  • Non-destructive technique
  • Macromolecular structure can be determined in solution, under wide range of ionic strength, pH (<7.4) and temperature conditions
  • Peptides with a maximal range of 3-4 kDa do not require isotope enrichment
  • NMR in solution can explore a wide range of macromolecular properties and not restricted to structure determination:
  • Dynamics
  • Folding
  • Interactions
  • Kinetics reactions in real time
  • Proteins in the range of 5-25 kDa require uniform labelling with 15N and 13C isotopes. Sometimes, partial deuteration is needed
  • Isotopes required for protein labelling are expensive and the cost increases as follow per L of media:
  • 15N (£) < 13C (£x8) < D2O (£x30)
  • De-novo protein synthesis is the only available method to incorporate isotopes into proteins and this is predominantly restricted to either E. coli or cell-free expression systems
  • High protein concentration is required (>250 µM) and it needs to be stable in solution for at least 24-48 h
  • Oligomeric and/or heterogenic samples are challenging
  • Data acquisition, processing, and analysis is relatively slow
  • An NMR spectrometer equipped with cryoprobe required for data collection
Advantages Disadvantages
  • Amenable to large macromolecular complexes
  • Can achieve near atomic resolution of proteins in their native (frozen hydrated) state
  • Multiple states of a macromolecule can be purified in silico from a single data set
  • Macromolecules viewed in real space, no phase problem
  • Viewing smaller proteins (<150 kDa) is challenging, size can be augmented with additional binding partners
  • Data acquisition, processing, and analysis is relatively slow
  • Computationally expensive (data storage and GPU)
  • Radiation damage – this can be mitigated to some degree
  • 200 kV or 300 kV electron microscope required for data collection

Protein quality is key

High-quality protein is paramount for a successful structural project. We invest in meticulous planning and apply our expertise to ensure the protein used in structural studies is of the highest possible quality, maximising the chances of success.

Our approach to structural biology projects

Structural projects are often best tackled with an iterative approach. We follow a milestone approach where at key points, data is discussed, and next steps agreed with our clients. The aim is to be guided by the data, to follow as efficient a pathway as possible and thereby reduce the risk.

Our service doesn’t just end with a final set of co-ordinates, but a detailed analysis of the 3D structure along with our insight and conclusions, driving ideas for project progression and drug design.