X-ray Crystallography: Exploring the Frontiers of Structural Biology
Article Jul 05, 2018 | By Dr Alison Halliday.
The fundamental approach to structure-based crystallography hasn’t really changed since 1913, when father and son duo, W. H. and W. L. Bragg, solved the first structure of any material at an atomic resolution.
“The basic experiment is to fire a beam of X-rays at a crystalline layer of whatever you’re studying, and you can calculate the structure of the molecule from the diffraction pattern,” explains Professor Brian Sutton, Professor of Molecular Biophysics at King’s College London.
Forty years later, images from the X-ray diffraction work of Rosalind Franklin enabled the discovery of one of the most famous molecular structures ever determined, Watson and Crick’s DNA double helix.
“We’ve always gone with the mantra of that it if you know the structure, you can understand how it works – that was certainly true for DNA,” says Sutton.
The field of X-ray crystallography is littered with Nobel prizes and continues to revolutionize our understanding of the structure of matter, with a wide impact spanning across physics, chemistry, biology and medicine.
More intense X-rays
As the technique is still highly dependent upon having a regular array of the material in 3-D, the biggest challenge still lies with getting crystals of a sufficient size and quality.
“Because biological molecules have so much inherent flexibility and heterogeneity, it can be very difficult to get good quality crystals,” explains Dr Andrew Leslie, Group Leader at the MRC Laboratory for Molecular Biology in Cambridge.
But powerful new X-ray sources, many orders of magnitude higher than can be achieved in a laboratory, are helping bypass this challenge. Nowadays, almost all structures are solved by using data collected at particle accelerators called synchrotrons, such as at the UK’s Diamond Light Source in Harwell, or the European Synchrotron Radiation Facility in Grenoble, France.
“They cut down on your data collection time if you’ve got large crystals, but you can also get data from much smaller crystals and that really has made a tremendous difference,” says Sutton.
The dramatic improvements in speed are also helping researchers to overcome heterogeneity in crystal quality.
“You might want to collect data from 30 crystals to find the best one and so to be able to do that in a realistic timescale you need to be able to collect data quickly,” explains Leslie.
Diffraction for destruction
More recently, X-ray free-electron lasers (XFELs) that can generate extremely intense pulses in the X-ray regime, are providing new opportunities for resolving sub-microscopic crystals.
“The idea of these new sources is to hit the crystal with a femtosecond X-ray pulse that’s so intense that it blows the crystal apart,” explains Leslie.
However, this destructive approach means it’s possible to generate only a single image for each crystal. But to complete a structural analysis, hundreds, or very often tens of thousands of diffraction images are required.
“So people are collecting hundreds of thousands of images by flowing a stream of crystals in a jet through the X-ray beam and collecting just one image from each,” says Leslie.
In 2017, scientists first demonstrated the huge potential of using this approach by solving the structure of the bovine enterovirus (BEV2).
Making nanoscale molecular movies
These high-energy sources, along with raising the temperature normally used for X-ray diffraction, are opening a new era of time-resolved structural studies.
Traditionally, structural biologists have focused their attention on collecting data from a single crystal in all possible rotations, using cryogenic temperatures to limit damage from the X-ray beam. However, this approach only provides a static picture.
“In the old days, your protein was stuck in a crystal in one orientation but in reality, it is probably pulsating,” explains Sutton.
Carrying out serial analyses at room temperature and/or using XFELs, opens the door to watching these subtle molecular motions that may be crucial to function. It also offers the exciting prospect of visualizing all the stages of an interactive process such as an enzyme during the catalytic cycle, or an antibody binding to an antigen.
“If you could have that going on while the crystals were streaming through the beam you could put all the snapshots of the individual stages of the process together and make a movie,” explains Sutton.
Scientists are undertaking these sorts of exciting studies right now.
“We really couldn’t do this before, but now we can – you can look at the processes of atoms moving just fractions of angstroms or nanometers,” enthuses Sutton.
Model-making with the help of computers
Advances in data collection, analysis and storage are also enabling researchers to study more complex biological structures, using sophisticated software and graphics programs that will automatically build their initial model.
“People can even get an initial structure while still at the synchrotron collecting the data,” says Leslie.
However, the first attempt is never perfect and will always need further refinement followed by careful validation.
“It’s possible for this refinement to distort it, so we then need to check that the final model has the expected stereochemistry,” explains Leslie.
Researchers will then deposit the final coordinates into the Protein Data Bank (PDB), a central repository with information on the 3-D shapes of proteins, macromolecules and complex assemblies, which now contains over 140,000 structures. The Cambridge Structural Database, which contains over 900,000 entries, provides a similar resource for small molecules.
“They run their own validation tools against the model and highlight anything that they think is unusual or possibly wrong and ask you to go back and take a closer look,” explains Leslie.
Nowadays, in the most straightforward cases, the whole process of creating crystals to getting a final structure can be done within a few weeks.
Structure-based drug design
The blue-skies objective of X-ray crystallography is to get information about the function of a molecule from its structure. But its most obvious practical application is in drug design, with many notable successes exemplifying its power, including drugs for HIV and cancer.
G-protein coupled receptors (GPCRs) are a family of transmembrane signalling proteins that are of crucial importance in pharmacology due to their substantial involvement in different diseases, with an estimated one-third of all drugs targeting them. As many have off-target effects, researchers have been trying to deduce the structure of many GPCRs to aid the design of more specific drugs with fewer side-effects. However, for many years they have proved extremely difficult to crack as they are unstable and very difficult to crystallize.
“People have been working on these for 30 years or more and getting nowhere, but over the last 10 years or so there’s been an explosion in the number of GPCRs that have been structurally determined,” enthuses Leslie.
As well as teaching biologists more about how GPCRs work, this flurry of structural information is also fueling drug discovery.
“There are a number of compounds now in various stages of clinical trials that have been based on this structural information from crystallography,” says Leslie.
Studying nature in its tiniest detail
Over a century since its inception, X-ray crystallography continues to make a huge impact across many areas of biology and medicine – but the basic approach remains the same.
“That’s the amazing thing, even with all the new technologies, you’re still basically just firing X-rays at ordered arrays of molecules and working out from the diffraction pattern where the atoms are,” says Sutton.
The biggest challenge remains around generating the crystals, but technological advances are enabling researchers to add to their growing catalogue of structures and watch tiny molecular movements that may be crucial to function.
It’s not hard to appreciate why those working in the field are so passionate about their work.
“I love seeing structures for the first time, it’s a wonderful feeling,” enthuses Sutton.
The rate at which new drugs are discovered is in decline, and computer-aided drug design has not produced the radical change in the success of candidate drugs the pharma industry anticipated. Artificial intelligence (AI)-based approaches are the new hope for revolutionizing the drug discovery process. But are these, as computer-aided drug design was, an over-hyped time bomb waiting to be found out? Or is it truly a revolution on the cusp of realizing its promise?READ MORE