Russell Fung

Trajectories of the ribosome as a Brownian nanomachine

Significance Many functions in the cell are performed by Brownian machines, macromolecular assemblies that use energy from the thermal environment for many of the conformational changes involved in their work cycles. Here we present a new approach capable of mapping the continuous motions of such nanomachines along their trajectories in the free-energy landscape and demonstrate this capability in the context of experimental cryogenic electron microscope snapshots of the ribosome, the nanomachine responsible for protein synthesis in all living organisms. We believe our approach constitutes a universal platform for the analysis of free-energy landscapes and conformational motions of molecular nanomachines and their dependencies on temperature, buffer conditions, and regulatory factors. A Brownian machine, a tiny device buffeted by the random motions of molecules in the environment, is capable of exploiting these thermal motions for many of the conformational changes in its work cycle. Such machines are now thought to be ubiquitous, with the ribosome, a molecular machine responsible for protein synthesis, increasingly regarded as prototypical. Here we present a new analytical approach capable of determining the free-energy landscape and the continuous trajectories of molecular machines from a large number of snapshots obtained by cryogenic electron microscopy. We demonstrate this approach in the context of experimental cryogenic electron microscope images of a large ensemble of nontranslating ribosomes purified from yeast cells. The free-energy landscape is seen to contain a closed path of low energy, along which the ribosome exhibits conformational changes known to be associated with the elongation cycle. Our approach allows model-free quantitative analysis of the degrees of freedom and the energy landscape underlying continuous conformational changes in nanomachines, including those important for biological function.

High-resolution structure of viruses from random diffraction snapshots

The advent of the X-ray free-electron laser (XFEL) has made it possible to record diffraction snapshots of biological entities injected into the X-ray beam before the onset of radiation damage. Algorithmic means must then be used to determine the snapshot orientations and thence the three-dimensional structure of the object. Existing Bayesian approaches are limited in reconstruction resolution typically to 1/10 of the object diameter, with the computational expense increasing as the eighth power of the ratio of diameter to resolution. We present an approach capable of exploiting object symmetries to recover three-dimensional structure to high resolution, and thus reconstruct the structure of the satellite tobacco necrosis virus to atomic level. Our approach offers the highest reconstruction resolution for XFEL snapshots to date and provides a potentially powerful alternative route for analysis of data from crystalline and nano-crystalline objects.

Conformations of macromolecules and their complexes from heterogeneous datasets

We describe a new generation of algorithms capable of mapping the structure and conformations of macromolecules and their complexes from large ensembles of heterogeneous snapshots, and demonstrate the feasibility of determining both discrete and continuous macromolecular conformational spectra. These algorithms naturally incorporate conformational heterogeneity without resort to sorting and classification, or prior knowledge of the type of heterogeneity present. They are applicable to single-particle diffraction and image datasets produced by X-ray lasers and cryo-electron microscopy, respectively, and particularly suitable for systems not easily amenable to purification or crystallization.

Dynamics from noisy data with extreme timing uncertainty

Imperfect knowledge of the times at which ‘snapshots’ of a system are recorded degrades our ability to recover dynamical information, and can scramble the sequence of events. In X-ray free-electron lasers, for example, the uncertainty—the so-called timing jitter—between the arrival of an optical trigger (‘pump’) pulse and a probing X-ray pulse can exceed the length of the X-ray pulse by up to two orders of magnitude, marring the otherwise precise time-resolution capabilities of this class of instruments. The widespread notion that little dynamical information is available on timescales shorter than the timing uncertainty has led to various hardware schemes to reduce timing uncertainty. These schemes are expensive, tend to be specific to one experimental approach and cannot be used when the record was created under ill-defined or uncontrolled conditions such as during geological events. Here we present a data-analytical approach, based on singular-value decomposition and nonlinear Laplacian spectral analysis, that can recover the history and dynamics of a system from a dense collection of noisy snapshots spanning a sufficiently large multiple of the timing uncertainty. The power of the algorithm is demonstrated by extracting the underlying dynamics on the few-femtosecond timescale from noisy experimental X-ray free-electron laser data recorded with 300-femtosecond timing uncertainty. Using a noisy dataset from a pump-probe experiment on the Coulomb explosion of nitrogen molecules, our analysis reveals vibrational wave-packets consisting of components with periods as short as 15 femtoseconds, as well as more rapid changes, which have yet to be fully explored. Our approach can potentially be applied whenever dynamical or historical information is tainted by timing uncertainty.

Conformational landscape of a virus by single-particle X-ray scattering

Using a manifold-based analysis of experimental diffraction snapshots from an X-ray free electron laser, we determine the three-dimensional structure and conformational landscape of the PR772 virus to a detector-limited resolution of 9 nm. Our results indicate that a single conformational coordinate controls reorganization of the genome, growth of a tubular structure from a portal vertex and release of the genome. These results demonstrate that single-particle X-ray scattering has the potential to shed light on key biological processes.

Single-particle structure determination by X-ray free-electron lasers: Possibilities and challenges

Single-particle structure recovery without crystals or radiation damage is a revolutionary possibility offered by X-ray free-electron lasers, but it involves formidable experimental and data-analytical challenges. Many of these difficulties were encountered during the development of cryogenic electron microscopy of biological systems. Electron microscopy of biological entities has now reached a spatial resolution of about 0.3 nm, with a rapidly emerging capability to map discrete and continuous conformational changes and the energy landscapes of biomolecular machines. Nonetheless, single-particle imaging by X-ray free-electron lasers remains important for a range of applications, including the study of large “electron-opaque” objects and time-resolved examination of key biological processes at physiological temperatures. After summarizing the state of the art in the study of structure and conformations by cryogenic electron microscopy, we identify the primary opportunities and challenges facing X-ray-based single-particle approaches, and possible means for circumventing them.

Determination of two-photon excitation and emission spectra of fluorescent molecules in single living cells

Modelocked Ti:Sapphire lasers are widely used in two-photon microscopes (TPM), partly due to their tunability over a broad range of wavelengths (between 700 nm and 1000 nm). Many biophysical applications, including quantitative Förster Resonance Energy Transfer (FRET) and photoswitching of fluorescent proteins between dark and bright states, require wavelength tuning without optical realignment, which is not easily done in tunable Ti:Sapphire lasers. In addition, for studies of dynamics in biological systems the time required for tuning the excitation should be commensurate with the shortest of the time scales of the processes investigated. A set-up in which a modelocked Ti:Sapphire oscillator providing broad-bandwidth (i.e., short) pulses with fixed center wavelength is coupled to a pulse shaper incorporating a spatial light modulator placed at the Fourier plane of a zero-dispersion two-grating setup, represents a faster alternative to the tunable laser. A pulse shaping system and a TPM with spectral resolution allowed us to acquire two-photon excitation and emission spectra of fluorescent molecules in single living cells. Such spectra may be exploited for mapping intracellular pH and for quantitative studies of protein localization and interactions in vivo.

Surface structure solution by X-ray diffraction: structure completion with positivity and atomicity constraints

We propose a method for the solution of surface structures from X-ray diffraction based on the phasing of measured structure factor amplitudes in two stages. First, incorporation of calculated amplitudes and phases of the structure factors of the known underlying bulk structure, together with an iterative algorithm that ensures the positivity of the electron density enables the determination of the amplitudes and phases of the surface structure factors contributing to the crystal truncation rods. Second, inclusion of these structure factors in an iterative algorithm based on Sayres equations enables the phasing of the superstructure rods.

Oiling the gate: a mobile application to improve the admissions process from the emergency department to an academic community hospital inpatient medicine service

ABSTRACT The process of admitting patients from the emergency department (ED) to an academic internal medicine (AIM) service in a community teaching hospital is one fraught with variability and disorder. This results in an inconsistent volume of patients admitted to academic versus private hospitalist services and results in frustration of both ED and AIM clinicians. We postulated that implementation of a mobile application (app) would improve provider satisfaction and increase admissions to the academic service. The app was designed and implemented to be easily accessible to ED physicians, regularly updated by academic residents on call, and a real-time source of the number of open AIM admission spots. We found a significant improvement in ED and AIM provider satisfaction with the admission process. There was also a significant increase in admissions to the AIM service after implementation of the app. We submit that the implementation of a mobile app is a viable, cost-efficient, and effective method to streamline the admission process from the ED to AIM services at community-based hospitals.

Sub-cycle strong-field influences in x-ray photoionization

We report the first evidence for strong field laser effects in x-ray ionization at LCLS. Experiments using a strong field optical-frequency laser and x-rays have revealed asymmetries in N2 fragmentation patterns that indicate sub-cycle dynamics.