THEME 2 SINGLE MOLECULE IMAGING

Bond cleavage of disulphide bridge in lysozyme induced by a femtosecond X-ray pulse from a free-electron laser. Observations of this phenomenon by the group of ISAC member Professor Ilme Schlichting (Heidelberg) led to the development of plasma-based molecular dynamics algorithms within the Imaging CoE (Image credit Alexander Kozlov).

“To image single biological molecules, we need a new breed of lenses that can focus powerful pulses of x-rays to a spot the same size as these molecules. Breakthroughs in the design, manufacture, diagnosis and imaging methods are required to bring these new lenses to life.”

Single molecule imaging reveals biologically relevant heterogeneities within proteins and protein complexes. Rather than producing an average or dominant structure of a protein or complex, single molecule techniques can capture the ‘heterogeneity landscape’ occupied by a single particle. The details of this landscape convey information about biochemical reactions and the functionality of biomolecules within living systems.

PROF. HARRY QUINEY

AT A GLANCE

Imaging CoE scientists perform single particle experiments that encompass X-ray free-electron laser single particle imaging (XFEL-SPI), cryogenic electron microscopy (cryo-EM), micro electron diffraction (micro-ED) and fluorescence imaging. Each of these techniques faces different and significant challenges in order to achieve single molecule imaging. It is not enough merely to achieve atomic scale molecular imaging, because this has no biological value if the molecule is not in its native state. The real challenge is to adapt and develop our techniques to better accommodate the intrinsic inhomogeneity of the systems we wish to image.

Imaging CoE researchers are advancing this field by combining XFEL-SPI or cryo-EM studies with complementary experiments using crystallography and molecular fluorescence. Such advances can come only through a collaboration spanning physics, chemistry and biology and involving both experiment and theory to determine the real behaviour of the target molecules under imaging conditions.

Our Centre’s expertise in single-molecule microscopy at UNSW, and a technology developed at La Trobe University to produce nano-droplets on demand, will allow us to devise protocols for the appropriate handling of samples in XFEL experiments, delivering molecules in their native states.

We have engaged with experimental work performed by Professor Ilme Schlichting, a member of our International Scientific Advisory Committee, on the effects of electronic damage on pump-probe XFEL measurements of molecular dynamics. We developed closer links with the Hamburg-based CFEL group led by Professor Henry Chapman (Imaging CoE Partner Investigator) and recently recruited Dr Andrew Morgan from Hamburg to join the Melbourne node of the Centre

ACTIVITY PLAN

  1. Utilise topological data analysis to establish the “shape” of large, noisy and incomplete data sets in cryo-EM, XFEL-SPI,
    including the effects of damage on the efficiency of the imaging systems.
  2. Explore electronic damage processes in XFEL imaging techniques.
  3. Develop new sample delivery systems to improve the outcomes of XFEL-SPI experiments.
  4. Explore micro electron diffraction techniques using the Imaging CoE’s expertise across both theoretical analysis
    and experimental studies.

HIGHLIGHT

New x-ray lenses focus on single molecule imaging

For decades the potential for Free-Electron Lasers (FELs) to offer damage free, time and conformationally resolved images of single biological molecules in their native state has been known. This was one of the major motivating factors for their construction. Until recently, however, scientific studies using FELs have focused on imaging large molecular ensembles, in the form of crystals, solid or liquid state materials or gases.

At the Imaging CoE, scientists are tackling this problem from two directions. Firstly, in collaboration with the SPI initiatives at the LCLS and European XFEL, new analysis methods are being developed that will be able to determine the electronic structure of a sample even in the low signal limit [1]. Secondly, in partnership with Dr Saša Bajt and Prof. Henry Chapman, both group leaders in Hamburg Germany, new lenses are being developed and tested that can focus high-energy short pulses of radiation to a spot of less than 10 nm in diameter.

Dr Bajt’s work on fabricating wedged multilayer Laue lenses has already resulted in record breaking focusing efficiencies (see Figure A). Wedging each bi-layer of the lens precisely ensures that each X-ray satisfies the ‘Bragg scattering condition’ and is focused so that almost no power is lost in the beam. However, pushing the boundaries of such a design is incredibly challenging. Errors in the placement of a layer by only a few atomic lengths can measurably disrupt the lens focus and efficiency.

Scientists at the Imaging CoE have been developing new tools to analyse the lens performance and determine the fabrication steps necessary to correct for the observed errors. This new tool, ‘ptychographic x-ray speckle tracking’, is essential to achieve a rapid test and design development cycle.

This work is not only a critical step towards the ultimate goal of acquiring ‘molecular movies’ of small bio-molecules at FEL facilities (including parthenogenic bacteria and viruses), but also to provide images of nano-scale structures using high energy X-rays at synchrotron facilities around the world – such as the Australian Synchrotron located at Monash University [2-4].

PAPERS:

1. Low-signal limit of X-ray single particle diffractive imaging. Vol 27, pp: 37816-37833 (2019), Kartik Ayyer et al., Opt. Express.

2. Ptychographic X-ray Speckle Tracking with Multi-Layer Laue Lens Systems (under review – 2019), Andrew Morgan et al., Journal of Applied Crystallography, special issue on ptychography.

3. Ptychographic X-ray Speckle Tracking (under review – 2019), Andrew Morgan et al., Journal of Applied Crystallography, special issue on ptychography.

4. Speckle-tracking: a Software Suite for Ptychographic X-ray Speckle Tracking (under review – 2020), Andrew Morgan et al., Journal of Applied Crystallography, special issue on ptychography.

Ray-tracing model of multi-layer lens design.

ACHIEVEMENT

Plasma formation and molecular dynamics in XFEL imaging

The use of femtosecond XFEL pulses for imaging molecular structure and dynamics induces secondary processes, such as photoionization, Auger decay and electron recapture that influence the information that is captured in diffraction experiments. Data obtained by the group from Imaging CoE ISAC member, Professor Ilme Schlichting, indicated that the ionization dynamics of proteins favours the preferential cleaving of disulphide bonds early in the imaging process (see figure).

This led the Theory Group to develop a new model for representing these dynamical systems in which the electrons are described using plasma physics and the nuclear motion using standard classical techniques. This hybrid scheme is in good agreement with more computationally intensive approaches in which the electron dynamics is described by an ensemble average of classical trajectories, determined by computationally intensive methods.

The two approaches are complementary, since the more computationally intensive scheme may be used to calibrate the underlying model parameters of the plasma and, in particular, the redistribution of energy through collisional processes. This new approach is under active development and has been used to analyse experimental data obtained in femtosecond pump-probe experiments. These theoretical and experimental methods are providing an essential window on the dynamics of biomolecules under XFEL imaging conditions.

Bond cleavage of disulphide bridge in lysozyme induced by a femtosecond X-ray pulse from a free-electron laser. Observations of this phenomenon by the group of ISAC member Professor Ilme Schlichting (Heidelberg) led to the development of plasma-based molecular dynamics algorithms within the Imaging CoE (Image credit Alexander Kozlov).
Diffraction tomography in electron diffraction

This group has investigated multiple electron scattering and in-molecule free-space propagation in transmission electron microscopy (EM), including cryo-EM, of small molecules. We have found that in-molecule Fresnel diffraction (free-space propagation) is significant due to the shallow depth of focus under the experimental conditions typical for cryo-EM. As a consequence, the projection approximation, on which the conventional computed tomography (CT) is based, is of diminished validity in high-resolution EM. A more suitable reconstruction method in this context is represented by diffraction tomography (DT) which can produce more accurate reconstruction of three-dimensional structure. We have proposed a new simplified DT method and tested it on numerically simulated examples (Figure B).

The group has also developed a practical method for unambiguous determination of the types and positions of atoms in small molecules from defocus series. This method has been named Pattern Matching Tomography (PMT) and is similar in its general approach to Big Bang Tomography proposed by Dirk van Dyck and co-authors in 2012. We have carried out numerical tests of the PMT method using multi-slice calculations of defocus series and CT scans of several biological molecules. The technique will be further developed to utilize Bayesian or machine-learning approaches in the context of cryo-EM

Figure B: Simulated EM images of lasso peptide 3NJW molecular structure obtained under the conditions of plane monochromatic electron wave illumination, refocused to the central plane of the molecule: (a) “ideal” image; (b) image corresponding to objective aperture of 70 mrad, thermal vibrations with 0.1 Å root-mean-square displacement and 1% Poisson shot noise. This example demonstrates the capabilities of the multislice-based EM simulation software that we have developed.