Methods and Tools for crystallography and cryo-em sample preparation

Conference: 2020: 70th ACA Annual Meeting
08/07/2020: 12:00 PM  - 3:00 PM 
4.1.1 
Education Session 
Virtual  

Description

With the technological advancements in both X-ray crystallography and Cryo-EM, structural biology techniques are becoming readily accessible to all labs. Although we are witnessing many significant strides in this field, the main bottleneck for both methods is still the preparation of high-quality protein samples. In this session we will highlight the latest methods and techniques for protein sample preparation for both crystallography and Cryo-EM experiments.

Presentations

Stanford-SLAC Cryo-EM Center

12:00 PM - 12:30 PM 
Stanford-SLAC Cryo-EM Center is a NIH funded resource which has the two missions: (1) to provide the scientific community access to state-of-the-art cryo-EM instruments for data collection towards atomic resolution structure determination of biochemically purified single particles and (2) to enable scientists across the nation to become independent cryo-EM investigators. Our Center also has cryo-specimen preparation devices, data quality assessment tools for on-the-fly evaluation and visualization, and image data storage capacity on a short-term basis. We provide expert staff that trains, assists and advises users, both on-site and remotely, and are available for consultation beforehand and in a follow-up program. We cross-train scientists who want to employ cryo-EM within their own research portfolios. Our training is targeted at a wide variety of skill levels - including short-term and long-term in-residence training programs for single particle cryo-EM workflow from specimen preparation to structure validation. Access to the Center is through an open process based on scientific merit. Acknowledgement: This research is supported by National Institutes of Health Common Fund Transformative High Resolution Cryo-Electron Microscopy program. 

View Proposal 422

Author

Wah Chiu Menlo Park, CA 

Online Crystallography: Fully Automated, Remote Controlled Protein-to-Structure Pipelines

12:30 PM - 1:00 PM 
The systematic introduction of automation in structural biology over the last decade has enabled the study of ever more challenging targets, and facilitated the use of crystallography in drug design. However, manual crystal mounting and processing methods are insufficient to exploit the full potential of modern synchrotron facilities. We have developed a novel approach called CrystalDirect that enables fully automated crystal mounting and cryo-cooling closing the automation gap between crystallization and X-ray data collection. The CrystalDirect technology also allows the automated delivery of small molecules to crystals, giving access to large scale small molecule screening through X-ray crystallography. We have combined this approach with the Massively Automated Sample Selection Integrated Facility (MASSIF) at ESRF to develop a fully automated, remote-controlled pipelines for macromolecular crystallography and an automated pipeline for large scale compound and fragment screening to support structure guided drug discovery programs. In order to facilitate high throughput data analysis we have built a series of Application Program Interfaces (APIs) linking the Crystallization Information Management System (CRIMS) and the ISPyB system for automated synchrotron data collection with automated structure refinement and analysis. These pipelines effectively provide online access to crystallization and synchrotron diffraction and data analysis facilities and remove key bottlenecks in modern crystallography. They can contribute to the rapid progression of challenging projects in structural biology, to facilitate the access to protein crystallography for scientist of other disciplines and stimulate translation of basic research into biomedical applications. On the other hand, the large amounts of data generated pose new challenges, but also provide new opportunities to develop integrated systems for data acquisition, processing and analysis, that facilitate the incorporation of data available in biological databanks into the analysis process. The experience from the use of these pipelines as well as the new opportunities enabled by the integration of crystallization, X-ray data collection and analysis into continuous, fully automated workflows will be discussed. 

View Proposal 439

Author

Jose Marquez, European Molecular Biology Laboratory, Grenoble Grenoble

Coffee Break

1:00 PM - 1:20 PM 

Complementing high resolution structure methods with small angle X-ray scattering data.

1:20 PM - 1:45 PM 
The last decades were accompanied by impressive advancements for many of the structural biology techniques. Classical approaches such as NMR and macromolecular X-ray crystallography have profited from the developments in high-field magnets and synchrotron radiation, the latter allowing to study microcrystals – even in vivo. Paired with advances in automation, high throughput screening of drug candidates from many therapeutic areas have become possible. The free electron lasers may further pave the way for the analysis of even smaller samples such as nanocrystals, nanoclusters and single molecules. Impressively, the "resolution revolution" in cryoEM has resulted in many intriguing new structures, now even with the possibility to detect single hydrogens. However, the bottleneck of these experiments remains the preparation of high quality samples. Protein crystallographers still have to spend a respectful amount of time performing trial-and-error approaches to obtain diffracting crystals. Even in the era of the "resolution revolution", electron microscopists need to master the process of preparing high-quality grids on which the homogenous macromolecules are evenly distributed in random orientations and embedded in vitreous thin films. And even after successful data collection and interpretation of the data, the question remains of the relevance of the obtained high resolution models for natural environments such as native-like solutions. Here, we will discuss the unique niche that small-angle X-ray scattering (SAXS) occupies and usefully complements such high resolution studies. One of SAXS main advantages is the ability to quantitatively characterize complicated systems and mixtures in native environments and their responses to changing physical and chemical conditions. SAXS can provide low resolution structures ab initio, validate available high resolution structures in solution environment and aid in constructing hybrid models utilizing partial models of domains or subunits. Furthermore, the degree of flexibility can be assessed providing, among others, also hints to why a probe is not crystallizing. Importantly, the sophisticated ensemble approaches and mixture analysis can be used to address the question of sample polydispersity. The presented examples will include current investigations on disease-related antibodies and biomedical relevant molecules such as the oligomerization states of Insulin formulations and Sars-CoV-2 Spike S1 protein. 

View Proposal 326

Author

Melissa Graewert, EMBL Hamburg Hamburg

Additional Author

Dmitri Svergun, EMBL Hamburg Hamburg

A Portable Fixed-Target Sample Delivery System for in-situ Serial Crystallography

1:45 PM - 2:10 PM 
Protein crystallography enables atomic-level structure determination from crystalline specimens. Due to the rapid onset of radiation damage at room temperature, the vast majority of protein crystal structures are done at cryogenic temperatures. While cryocooling alleviates much of the practical issues of radiation damage, it also restricts the energy landscapes in which proteins normally exist, which can lead to inaccurate pictures on the room temperature dynamics of conformation, allostery, and substrate binding. We have developed a scalable, portable fixed-target delivery system for serial crystallography that enables rapid data collection with minimized background. Ultrathin Kapton platforms are photopatterned to create wells that can localize crystals into a regular grid, and remaining mother liquor can be removed by vacuum or wicking with a paper towel to minimize background. For sensitive or difficult crystals, these platforms are also highly amenable to in-situ crystallization methods. The platform design allows crystal growth to be localized around well spaces, which simplifies data collection by rastering. We have demonstrated the utility of our new design on a number of novel drug-binding proteins. 

View Proposal 293

Author

Aaron Finke, Cornell University Groton, NY 

Additional Author(s)

Gabrielle Illava, Cornell Ithaca, NY 
Benjamin Apker, MiTeGen
Richard Jayne, MiTeGen, Inc. Ithaca, NY 
David Closs, MiTeGen, Inc. Ithaca, NY 
Qingqiu Huang, Cornell University Ithaca, NY 
Irina Kriksunov, Cornell University Ithaca, NY 
Shawn Milano, Cornell University Ithaca, NY 
David Schuller, MacCHESS, Cornell Univ Ithaca, NY 
Doletha Szebenyi, MacCHESS, Cornell Univ Ithaca, NY 
Richard Cerion, Cornell University Ithaca, NY 
Robert Thorne, Physics Dept, Cornell Univ

Through-grid wicking enables high-speed cryoEM specimen preparation

2:10 PM - 2:35 PM 
Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. We show that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass fiber filter held against the opposite side, often called the 'back' of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. We combined ultrasonic specimen application and through-grid wicking in a high-speed specimen preparation device that we name "Back-it-up", or BIU. The high liquid-absorption capacity of the glass fiber compared to self-wicking grids appears to make the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice suitable for cryoEM, while the device's speed reduces adoption of a preferred orientation in a test specimen. (Preprint available: https://www.biorxiv.org/content/10.1101/2020.05.03.075366v1.full) 

View Proposal 285

Author

Yong Zi Tan

Additional Author

John Rubinstein, The Hospital for Sick Children Toronto, Ontario 

Effects of chameleon dispense-to-plunge speed on particle concentration, complex formation, and final resolution: A case study using a bacterial ribonucleotide reductase

2:35 PM - 3:00 PM 
The chameleon (SPT Labtech; based on the NYSBC SpotItOn) utilizes a picoliter spraying method to dispense a thin stripe of sample onto self-wicking cryo-electron microscopy (cryo-EM) grids. This method allows for extremely fast (as fast as 54 ms) dispense-to-plunge (D2P) times. The fast D2P timing, combined with the unique physics of spraying and wicking, has opened a new pathway for difficult samples that display preferred orientation or complex denaturation at the air/water interface. Here, we present a case study utilizing the chameleon to ameliorate the effects of complex denaturation through faster D2P times. Using a 528-kDa bacterial ribonucleotide reductase complex, we demonstrate improved complex stability and final resolution using the chameleon at four different D2P times (619 – 54 ms) as well as trends in protein concentration for varying D2P speeds. 

View Proposal 440

Author

Talya Levitz

Additional Author(s)

Catherine Drennan, MIT
Paul Thaw, SPT Labtech Royston, Herts