How can we more easily produce high quality protein crystals to be used for x-ray crystallography for protein structure identification?

Hi! My name is Josh Werblin and I am a rising junior at Northwestern University studying biomedical engineering. This summer, I have been working in Gyorgy Babnigg’s lab, investigating the applicability of droplet-based microfluidics in obtaining high-quality protein crystals.

He is part of the Midwest Center for Structural Genomics (MCSG, PI: Andrzej Joachimiak) and the Center for Structural Genomics of Infectious Diseases (CSGID, co-directors: Karla Satchell, Andrzej Joachimiak), two structural biology efforts that offer atomic-level insights into the function of proteins and their complexes.  Currently, high-quality protein crystals are difficult to generate, and account for one of the major bottlenecks in structural biology. During my summer project, I tested the feasibility of using droplet-based microfluidics to generate crystals amenable for structural studies.

Generation of protein droplets GIF
Slowed footage of droplets being generated. The two channels from the left are the reagent channels, protein and crystallization screen, respectively. The vertical channels are oil that pinches off droplets of the reagents.

To get crystals to form, you need to combine a purified protein with crystallization screens, which is a set of 96 mix of buffers, salts, and precipitants. Unfortunately, finding the right crystallization condition to make crystals for a protein can take a lot of testing. Even when you figure out a compatible crystallization after testing many crystallization screens and incubation conditions for a protein that yields crystals, the resulting crystals are too tiny for data collection, only a few microns across. Droplet-based microfluidics has the potential to test the many combinations using only small amount of protein for testing.

I tested the crystallization of a previously characterized protein using the microfluidic setting. Small aqueous droplets are formed in fluorinated oil. This can be done by using a small chip and pumping your reagents in. This stream of protein and crystallization screen are cut off by two streams of oil, which creates the droplets.

Using droplet-based microfluidics, I generated tiny droplets containing the protein and crystallization screen at the right concentrations, and was able to grow relatively large crystals (over 50 microns).

The protein I’ve focused on this summer is an enzyme called sialate O-acetylesterase from one of the good bacteria in our gut (Bacteroides vulgatus). After testing different concentrations and conditions, I finally generated some really good-looking crystals!

Two images of droplets showing multiple different sized crystals
Left: Small variations in the ratio of protein and crystallization solution result in droplets with single large crystals, a few smaller crystals, or many microcrystals.
Right: An image of big protein crystals ready to be put onto the beam-line for x-ray crystallography.

With everything all ready, Gyorgy and his colleague, Youngchang Kim, were able to test these crystals at the SBC beam-line of the Advanced Photon Source and hopefully soon I will have the structure of this protein!

I am so thankful to have had this opportunity to work in this lab with wonderful and knowledgeable people and I learned so much about 3D design and printing, experimental design, and proteins and their crystallization.

Students present their summer’s work at Argonne

This morning the students delivered compelling presentations about their research in a diverse set of areas that have been highlighted on this blog.  They did an excellent job communicating the complexities of their work to an audience made up of technical experts in a range of disciplines.  This event truly showcased their talents as researchers and communicators.   

Ugly Boxes + Experimental Sensors

Hello! I’m Jordan Fleming, a recent graduate of Northwestern University’s Mechanical Engineering and Environmental Engineering departments. I’ll soon be starting my Master’s with a focus in Water and Energy Engineering. I’ve spent this summer working on the Waggle sensor platform for Ugly Boxes and Ugly Kits (yes, that’s really what they’re called, and yes, they could stand to be slightly more attractive) with Peter Beckman and Rajesh Sankaran. Waggle is an integrated, intelligent, attentive sensor designed and developed at Argonne National Laboratory. The Waggle platform enables distributed sensing through edge computing and on-the-node data storage, a deviation from traditional sensors that simply collect data. While the Array of Things (AOT) node is the official design that is deployed in the city of Chicago, the Ugly Box allows for experimentation and development of both the core Waggle platform and also testing and integrating new sensors. One of the main advantages of the Ugly Boxes is that they are also conducive to use with smaller Arduino or embedded modules like Photon and Electron Particle boards, that can transfer important information via WiFi and cellular communication. The sensors are adaptable to fit the needs of the area and situation in which they are deployed. The nodes can gather data a wide variety of data from conventional environmental parameters like barometric pressure and sulfur dioxide concentrations, to computed inferences through computer vision and machine learning algorithms deployed on the nodes. This makes it possible for the nodes to detect standing water to signal flooding, sky color and cloud cover, and the number of pedestrians in an intersection, among others.

I’ve been streamlining Ugly Box manufacture, plugin creation, and sensor testing and documentation. I’ve enjoyed learning new things like coding in Python and C. The sensors I’m working on will be deployed at Indian Boundary Prarie and the Chicago Botanic Gardens. I’m also working on sensors for Center for Neighborhood Technology’s (CNT) RainReady initiative to prepare ground and surface water level, and sump pump detection sensors in the basements of the homes of individuals living in the Chatham area looking to prevent residential flooding. Flooding is an important problem to address because it causes serious health problems and property damage. I’ve been testing sensor interfaces with the Ugly Box setup to ensure reliable quality in a variety of environmental conditions. In order to ensure these sensors can be used in homes, or anywhere really, I’ve been working on a Python script for publishing sensor data from Ugly Box, Photon, and Electron to the Waggle cloud, “Beehive”, a platform where data can be manipulated and analyzed. Creating an open-source system for sensor deployment will further the goal of extensibility, ease of use and adaptability. These endeavors align with the common principles of modularity in the intelligent, cloud computing, and urban-sensing Waggle platform.

The variety and quantity of data collected by the AoT nodes and Ugly Boxes have implications for policy improvements in many arenas, including public health, urban planning, urban heat insland effect quantification, and flood mapping. Additionally, groups like Chi Hack Night, composed of civically-focused students and professionals in the community who work with the City of Chicago’s open source data, will be better able to serve the community as a result of locality specific data. Capturing the pulse of the city through the AoT nodes and Ugly Boxes will increase Chicago’s operation efficiency, and improve the quality of life of its residents.

I’ve had so much fun this summer. Getting to know everyone in the office, meeting interns from other schools, and exploring different research career paths has been great. When the Waggle team isn’t working, you can find us at Wrigley field or an Indian buffet. Employees who dine together, stay together.

Waggle interns at Wrigley Field.
   Waggle interns at Wrigley Field.
Waggle Team enjoying lunch at an Indian buffet.

Fun and informative science communication workshop

Students are gearing up to present their work at a seminar this week and Michelle Paulsen and Byron Stewart Northwestern’s Ready Set Go (RSG) program that trains students in science communication visited Argonne to work with NAISE undergraduate researchers.

Students considered key elements of presentation preparation including knowing your audience, avoiding jargon, and framing the problem. They worked through some improvisations such as explaining to a partner, who posed as a time traveler, how and why to get through airport security.  This exercise relates to  explaining your research to a non-expert. They considered how to be persuasive as they convinced their cohorts to join them at a favorite lunch spot. And they practiced delivering their presentations one. word. at. a. time. They also received feedback on their upcoming presentations. We’re grateful to RSG for the visit and great insights and looking forward to student presentations this week at Argonne and early September at Northwestern. Students have the opportunity to showcase their valuable contributions and build bridges to collaborations between Argonne and Northwestern.

Growth of Ferromagnetic Cobalt Nanotubes using Atomic Layer Deposition

Hello everyone! My name is Braxton Cody, and next year I will be in my third year at Northwestern University studying Mechanical Engineering. This summer I have been working with Jonathan Emery of the Material Science Department at Northwestern University and Charudatta Phatak of the Material Science Division at Argonne National Laboratory to figure out how to create cylindrical nanotubes of Cobalt metal to characterize and the magnetic domain walls based on the curvature of the metal. To guarantee even coatings of Cobalt metal, atomic layer deposition (ALD) techniques will be used as a means to gain extremely precise control the thickness of the metal on the order of angstroms.

During the first part of this summer, I collected data regarding a specific ALD process for Cobalt recently developed by the Winter Group of Wayne State University. After establishing the specific processing parameters, becoming familiar with the various characterization techniques, and ordering the chemicals required for the ALD process, we began implanting the process resulting in varying degrees of success. We did manage to grow Cobalt metal as seen in the x-ray fluorescence data shown below. The data for the Pt/Co multilayer (shown in beige) was created using sputtering deposition as a standard for  comparison, providing information about the measurement sensitivity and accuracy.

Despite growing Cobalt metal, we soon discovered that a preparation error resulted in exposing the chemical to air ruining the precursor chemical. We ran a test to confirm our suspicions, and in the x-ray fluorescence data, there is no energy characteristic peak corresponding to Cobalt atoms, as seen in the figure below. Due to the expense of the chemical, we’ve begun pursuing alternate Co metal growth methods using plasma-enhanced ALD

Although we have encountered numerous setbacks, we will continue working towards controlled ALD of Cobalt. We are now researching new options involving Plasma-enhanced ALD as a cheaper and potentially more effective option. Due to the timeframe of this project, we hope to test this new chemistry on flat substrates and confirm Co metal deposition before the end of the summer. If that endeavor proves successful, we will proceed to grow the nanotubes and characterize their magnetic structures. While the previous weeks’ results have not proven successful, they have provided us with new directions for this project and set the groundwork that is instrumental in the continuation of this project.