Structure Determination Of Biological Macromolecules Research Articles

Metallo-supramolecular branched polymer protects particles from air-water interface in single-particle cryo-electron microscopy

Recent technological breakthroughs in single-particle cryo-electron microscopy (cryo-EM) enable rapid atomic structure determination of biological macromolecules. A major bottleneck in the current single particle cryo-EM pipeline is the preparation of good quality frozen cryo-EM grids, which is mostly a trial-and-error process. Among many issues, preferred particle orientation and sample damage by air–water interface (AWI) are common practical problems. Here we report a method of applying metallo-supramolecular branched polymer (MSBP) in the cryo-sample preparation for high-resolution single-particle cryo-EM. Our data shows that MSBP keeps a majority of particles away from air–water interface and mitigates preferred orientation as verified by the analyses of apoferritin, hemagglutinin) trimer and various sample proteins. The use of MSBP is a simple method to improve particle distribution for high-resolution structure determination in single-particle cryo-EM.

Ethoxylate Polymer-Based 96-Well Screen for Protein Crystallization

Crystallization is the limiting step in X-ray structure determination of biological macromolecules. As crystallization experiments can be largely automatized, the diversity of precipitant solutions is often the determinant factor to obtain crystals of high quality. Here, we introduce a 96-well screening kit of crystallization conditions, centered on three ethoxylate-based organic polymers as precipitants and various additional compounds to promote crystal formation. This crystallization screen was tested on various non-standard proteins from bacteria and archaea. Structure determination succeeded for seven out of thirteen targets based on crystals that frequently diffracted to a higher resolution than those obtained with commercially available screening kits. Crystallization hits were rarely similar among the three ethoxylate-based organic polymers and, in comparison, with already available crystallization screens. Hence, the presented crystallization screen is an efficient tool to complement other screens and increase the likelihood of growing crystals suitable for X-ray structure determination.

JINXED: just in time crystallization for easy structure determination of biological macromolecules

JINXED: just in time crystallization for easy structure determination of biological macromolecules

Dynamical scattering in ice-embedded proteins in conventional and scanning transmission electron microscopy.

Structure determination of biological macromolecules using cryogenic electron microscopy is based on applying the phase object (PO) assumption and the weak phase object (WPO) approximation to reconstruct the 3D potential density of the molecule. To enhance the understanding of image formation of protein complexes embedded in glass-like ice in a transmission electron microscope, this study addresses multiple scattering in tobacco mosaic virus (TMV) specimens. This includes the propagation inside the molecule while also accounting for the effect of structural noise. The atoms in biological macromolecules are light but are distributed over several nanometres. Commonly, PO and WPO approximations are used in most simulations and reconstruction models. Therefore, dynamical multislice simulations of TMV specimens embedded in glass-like ice were performed based on fully atomistic molecular-dynamics simulations. In the first part, the impact of multiple scattering is studied using different numbers of slices. In the second part, different sample thicknesses of the ice-embedded TMV are considered in terms of additional ice layers. It is found that single-slice models yield full frequency transfer up to a resolution of 2.5 Å, followed by attenuation up to 1.4 Å. Three slices are sufficient to reach an information transfer up to 1.0 Å. In the third part, ptychographic reconstructions based on scanning transmission electron microscopy (STEM) and single-slice models are compared with conventional TEM simulations. The ptychographic reconstructions do not need the deliberate introduction of aberrations, are capable of post-acquisition aberration correction and promise benefits for information transfer, especially at resolutions beyond 1.8 Å.

JINXED: just in time crystallization for easy structure determination of biological macromolecules.

Macromolecular crystallography is a well established method in the field of structural biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now under development towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand-soaking and cryo-protection. These handling steps can cause significant crystal damage, and hence reduce data quality. Furthermore, in time-resolved experiments based on serial crystallography, which use micrometre-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method that combines protein crystallization and data collection in a novel one-step process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg-white lysozyme and crystallization times of only a few seconds. This method, called JINXED (Just IN time Crystallization for Easy structure Determination), promises high-quality data due to the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches.

High-Resolution Cryo-Electron Microscopy Reveals the Unique Striated Hollow Structure of Photocatalytic Macrocyclic Polydiacetylene Nanotubes.

High-resolution structures are crucial for understanding the functional properties of nanomaterials. We applied single-particle cryo-electron microscopy (cryo-EM), a method traditionally used for structure determination of biological macromolecules, to obtain high-resolution structures of synthetic non-biological filaments formed by photopolymerization of macrocyclic diacetylene (MDA) amphiphilic monomers. Tomographic analysis showed that the MDA monomers self-assemble into hollow nanotubes upon dispersion in water. Single-particle analysis revealed tubes consisting of six pairs of covalently bonded filaments held together by hydrophobic interactions, where each filament is composed of macrocyclic rings stacked in parallel "chair" conformations. The hollow MDA nanotube structures we found may account for the efficient scavenging of amphiphilic pollutants in water and subsequent photodegradation of the guest species.

The FUSION protein crystallization screen.

The success and speed of atomic structure determination of biological macromolecules by X-ray crystallography depends critically on the availability of diffraction-quality crystals. However, the process of screening crystallization conditions often consumes large amounts of sample and time. An innovative protein crystallization screen formulation called FUSION has been developed to help with the production of useful crystals. The concept behind the formulation of FUSION was to combine the most efficient components from the three MORPHEUS screens into a single screen using a systematic approach. The resulting formulation integrates 96 unique combinations of crystallization additives. Most of these additives are small molecules and ions frequently found in crystal structures of the Protein Data Bank (PDB), where they bind proteins and complexes. The efficiency of FUSION is demonstrated by obtaining high yields of diffraction-quality crystals for seven different test proteins. In the process, two crystal forms not currently in the PDB for the proteins α-amylase and avidin were discovered.

Recent progress and future perspective of electron cryomicroscopy for structural life sciences.

The three-dimensional structure of biological macromolecules, such as proteins and nucleic acids, and their complexes is the fundamental information not only for life sciences but also for medical sciences and drug design. Electron cryomicroscopy has become an extremely powerful tool for high-resolution structural analysis of biological macromolecules, not just in addition to X-ray crystallography and nuclear magnetic resonance sepectroscopy (NMR) that have been used as the basic techniques in structural biology. By the development of hardware and software, such as transmission electron cryomicroscopes with highly stable and controllable electron optics, cold field emission gun and energy filter, complementary metal oxide semiconductor (CMOS)-based direct electron detectors with high frame rate and high sensitivity, high-speed computers and software programs for image analysis, electron cryomicroscopy now allows structure determination of biological macromolecules at atomic levels within a few days even from a drop of solution sample with an amount as small as a few micrograms. How can the structures of macromolecules be imaged and analyzed at atomic level resolution in their native states despite their high sensitivity to radiation damage at a relatively low level of electron irradiation? We describe recent progress and future perspective of electron cryomicroscopy for structural life sciences.

User capabilities at the GM/CA@APS Structural Biology Facility at the Advanced Photon Source

The General Medical Sciences and Cancer Institutes’ structural biology facility at the APS (GM/CA@APS) operates a National User Facility for crystallographic structure determination of biological macromolecules, with an emphasis on challenging, high-impact projects. The facility includes canted-undulator beamlines, 23ID-B and 23ID-D, that provide stable, intense X-ray beams of user-selectable size down to 5-micron, an intuitive user interface for experiment control, and an automated data processing pipeline.

Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes.

Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.

CASSPER is a semantic segmentation-based particle picking algorithm for single-particle cryo-electron microscopy

Particle identification and selection, which is a prerequisite for high-resolution structure determination of biological macromolecules via single-particle cryo-electron microscopy poses a major bottleneck for automating the steps of structure determination. Here, we present a generalized deep learning tool, CASSPER, for the automated detection and isolation of protein particles in transmission microscope images. This deep learning tool uses Semantic Segmentation and a collection of visually prepared training samples to capture the differences in the transmission intensities of protein, ice, carbon, and other impurities found in the micrograph. CASSPER is a semantic segmentation based method that does pixel-level classification and completely eliminates the need for manual particle picking. Integration of Contrast Limited Adaptive Histogram Equalization (CLAHE) in CASSPER enables high-fidelity particle detection in micrographs with variable ice thickness and contrast. A generalized CASSPER model works with high efficiency on unseen datasets and can potentially pick particles on-the-fly, enabling data processing automation.

Tidy up cryo-EM sample grids with 3D printed tools

Cryo-EM technology has developed to the point of high-throughput structure determination of biological macromolecules embedded in vitreous ice. Nonetheless, challenging targets need extensive sample screening, often of many cryo-EM sample grids prepared under various conditions. We have designed and made tools for manipulating sample grids in storage cases. These tools are made of a plastic fiber using a wide-use 3D printer, a fused deposition modeling type, and polished under acetone gas. A grid case stacker organizes many frozen-hydrated cryo-EM grids and the stackers can be piled up inside a standard 50 mL centrifuge tube. We have also introduced tools that facilitate handling of grid cases under liquid nitrogen and a stocker of the grid retainers contained in a CRYO ARM electron microscope. Blueprints of the tools named CryoGridTools are available from a GitHub site.

Methods for merging data sets in electron cryo-microscopy.

Recent developments have resulted in electron cryo-microscopy (cryo-EM) becoming a useful tool for the structure determination of biological macromolecules. For samples containing inherent flexibility, heterogeneity or preferred orientation, the collection of extensive cryo-EM data using several conditions and microscopes is often required. In such a scenario, merging cryo-EM data sets is advantageous because it allows improved three-dimensional reconstructions to be obtained. Since data sets are not always collected with the same pixel size, merging data can be challenging. Here, two methods to combine cryo-EM data are described. Both involve the calculation of a rescaling factor from independent data sets. The effects of errors in the scaling factor on the results of data merging are also estimated. The methods described here provide a guideline for cryo-EM users who wish to combine data sets from the same type of microscope and detector.

Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation.

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5-20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A2A adenosine receptor (A2AAR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A2AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A2AAR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5-20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.

BIODIFF: Diffractometer for large unit cells

The single crystal diffractometer BIODIFF, which is jointly operated by the Technische Universität München and JCNS, Forschungszentrum Jülich, is designed to handle crystals with large unit cells and is dedicated to the structure determination of biological macromolecules. The main field of application is the neutron structure analysis of proteins, especially the determination of hydrogen atom positions.

Structure calculation, refinement and validation using CcpNmr Analysis.

CcpNmr Analysis provides a streamlined pipeline for both NMR chemical shift assignment and structure determination of biological macromolecules. In addition, it encompasses tools to analyse the many additional experiments that make NMR such a pivotal technique for research into complex biological questions. This report describes how CcpNmr Analysis can seamlessly link together all of the tasks in the NMR structure-determination process. It details each of the stages from generating NMR restraints [distance, dihedral, hydrogen bonds and residual dipolar couplings (RDCs)], exporting these to and subsequently re-importing them from structure-calculation software (such as the programs CYANA or ARIA) and analysing and validating the results obtained from the structure calculation to, ultimately, the streamlined deposition of the completed assignments and the refined ensemble of structures into the PDBe repository. Until recently, such solution-structure determination by NMR has been quite a laborious task, requiring multiple stages and programs. However, with the new enhancements to CcpNmr Analysis described here, this process is now much more intuitive and efficient and less error-prone.

Relaxation-compensated difference spin diffusion NMR for detecting 13C-13C long-range correlations in proteins and polysaccharides.

The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly (13)C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular (13)C-(13)C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D (1)H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for (13)C T1 relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ~200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T1 relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T1 relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter-residue and intermolecular correlation peaks, which are often difficult to resolve in the parent 2D spectra.

Direct phase selection of initial phases from single-wavelength anomalous dispersion (SAD) for the improvement of electron density and ab initio structure determination.

Optimization of the initial phasing has been a decisive factor in the success of the subsequent electron-density modification, model building and structure determination of biological macromolecules using the single-wavelength anomalous dispersion (SAD) method. Two possible phase solutions (φ1 and φ2) generated from two symmetric phase triangles in the Harker construction for the SAD method cause the well known phase ambiguity. A novel direct phase-selection method utilizing the θ(DS) list as a criterion to select optimized phases φ(am) from φ1 or φ2 of a subset of reflections with a high percentage of correct phases to replace the corresponding initial SAD phases φ(SAD) has been developed. Based on this work, reflections with an angle θ(DS) in the range 35-145° are selected for an optimized improvement, where θ(DS) is the angle between the initial phase φ(SAD) and a preliminary density-modification (DM) phase φ(DM)(NHL). The results show that utilizing the additional direct phase-selection step prior to simple solvent flattening without phase combination using existing DM programs, such as RESOLVE or DM from CCP4, significantly improves the final phases in terms of increased correlation coefficients of electron-density maps and diminished mean phase errors. With the improved phases and density maps from the direct phase-selection method, the completeness of residues of protein molecules built with main chains and side chains is enhanced for efficient structure determination.

Developments in X-ray Crystallographic Structure Determination of Biological Macromolecules

The three-dimensional structures of large biomolecules important in the function and mechanistic pathways of all living systems and viruses can be determined by x-ray diffraction from crystals of these molecules and their complexes. This area of crystallography is continually expanding and evolving, and the introduction of new methods that use the latest technology is allowing the elucidation of ever larger and more complex biological systems, which are now becoming tractable to structure solution. This review looks back at what has been achieved and forward at how current and future developments may allow technical challenges to be overcome.

Reflections on the Many Facets of Protein Microcrystallography

The use of X-ray crystallography for the structure determination of biological macromolecules has experienced a steady expansion over the last 20 years with the Protein Data Bank growing from <1000 deposited structures in 1992 to >100 000 in 2014. The large number of structures determined each year not only reflects the impact of X-ray crystallography on many disciplines in the biological and medical fields but also its accessibility to non-expert laboratories. Thus protein crystallography is now largely a mainstream research technique and is routinely integrated in high-throughput pipelines such as structural genomics projects and structure-based drug design. Yet, significant frontiers remain that continuously require methodological developments. In particular, membrane proteins, large assemblies, and proteins from scarce natural sources still represent challenging targets for which obtaining the large diffracting crystals required for classical crystallography is often difficult. These limitations have fostered the emergence of microcrystallography, novel approaches in structural biology that collectively aim at determining structures from the smallest crystals. Here, we review the state of the art of macromolecular microcrystallography and recent progress achieved in this field.