Information encoded in our genes controls how we live and grow. As part of this complex process, DNA is transcribed to RNA, one "letter" (nucleotide) at a time, by an enzyme called RNA polymerase (RNAP). At a later stage, 3-nucleotide RNA sequences are translated into protein according to the genetic code.
From years of biochemical and structural studies, we have a good understanding of how transcription works - or do we? In "canonical transcription", each nucleotide (A, T, C, or G) in a DNA strand is transcribed into the single complementary nucleotide (U, A, G, or C) in RNA. However, as discovered by Nobel laureate Paul Berg in the 1960's, "reiterative transcription" can also occur, in which a single DNA nucleotide is transcribed into multiple copies of the complementary RNA nucleotide. The latter process, which has important regulatory roles in many species, is not so well understood, and recent work by Katsu Murakami and colleagues at Penn State provides some fascinating insight into how RNAP carries out this type of transcription.
Investigation of the mechanism of action of bacterial RNAP is a major project of the Murakami lab, and crystal structures determined using data from CHESS have been critical to the success of this work. Unlike many macromolecules, some RNAPs can function within a crystal, so that functional intermediate states can be observed. Previous results from the Murakami group have revealed many details of the canonical transcription process. Now, as reported in Proceedings of the National Academy of Sciences (Murakami et al. 2017, PNAS 114, 8211-8216), the group has determined a crystal structure for an RNAP complex caught in the act of reiterative transcription. Data were collected at the F1 station.
The structure (below) shows that, during reiterative transcription, the growing strand of RNA does not extend toward the RNA exit channel (as it does in the canonical case). Instead, it detours towards the "main channel" of RNAP, an action that allows RNA extension without shifting the DNA, so that multiple copies of a single nucleotide are made. The structure also allows determination of the unusual pathway that must be followed when reiterative transcription terminates and normal transcription of the rest of the DNA proceeds.
In the figure, RNA is shown in red, DNA in shades of green, and RNAP in gray (a subunit), white (ß' subunit), orange (s subunit), and blue (ß subunit, part of the domain omitted for clarity).
Submitted by: Marian Szebenyi, MacCHESS, Cornell University, 10/09/2017
Sol M. Gruner. Physics, College of Arts and Sciences
“Most scientists focus on a very specific area, but I do many different things,” says Sol Gruner, Physics. “I’m a research mutt. Mainly, I develop tools to attack scientific problems people haven’t looked at yet, largely because the tools needed to solve those problems haven’t existed.”
“Our goal is to examine what happens at the atomic level by shooting x-rays through the material as it is crushed,” says Gruner. “We want to break that microsecond process into bits…for each time bit, you’re getting an x-ray picture of the atomic structure as it evolves.”
Cornell Research. By Jackie Swift
RAW Power! MacCHESS software brings synchrotron-level data processing to the laptop and home laboratory
Since its introduction by Søren Skou (Nielsen) in 2010, the BioXTAS RAW software has been a familiar interface to the many biomedical scientists collecting data at CHESS beamlines in recent years. From the start, RAW was designed specifically with novice users in mind: when scientists arrive at the beamline, they need something fast and easy to learn in the very limited time available … often late at night. The program was literally designed by looking over the shoulders of beamline users as they collected data. But rather than simply create an automated data processing pipeline, we opted to give people the power to fully process data on their own computers at home, if they choose. This allows them to use the same software at other beamlines and even on their own home X-ray sources: from initial raw data reduction to final publication. Indeed, with over 4000 downloads in 2017, RAW is now the primary processing software at several other beamlines and lab source facilities worldwide.
Richard Gillilan, Jesse Hopkins, and Søren Skou at the annual American Crystallographic Association meeting where they conducted a tutorial in the BioXTAS RAW software. Their new paper describing the software will appear in the latest issue of Journal of Applied Crystallography (open access): scripts.iucr.org/cgi-bin/paper?ge5036
RAW is free, open source, and runs on Mac, Windows, and Linux. Jesse Hopkins (MacCHESS) has greatly expanded the capabilities and documentation of the program, making it easier than ever to install at home. RAW can read over 27 different detector image formats and has sophisticated masking and integration capabilities as well as a newly-added algorithm for statistically comparing and merging multiple exposures. Data can be placed on an absolute scale based on water or glassy carbon standards. The program can perform all the customary SAXS analyses such as Guinier analysis, Porod and normalized Kratky plots, and pair distance distribution functions. The program also can serve as an interface for several of the most popular algorithms in the widely used ATSAS suite for solution scattering developed at EMBL.
The most recent and exciting addition to RAW is its ability to untangle mixtures of biomolecules. RAW is specifically designed to work with size exclusion chromatography coupled SAXS (SEC-SAXS), a wildly popular new technique for separating and structurally analyzing mixtures of biomolecules. The software contains the first public implementation of the powerful new Evolving Factor Analysis (EFA) for extracting scattering curves from overlapping peaks in SEC-SAXS chromatograms. The method has already been used successfully by several early external users and promises to be a breakthrough in solving tough separation problems.
BioXTAS RAW software showing analysis of size exclusion chromatography coupled to small angle X-ray measurements (SEC-SAXS).
Full details about RAW including validation tests can be found in the latest issue of the Journal of Applied Crystallography: Hopkins. J. B., Gillilan, R. E., Skou, S. “BioXTAS RAW: improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis.” J. Appl. Cryst. (2017), 50. DOI: 10.1107/S1600576717011438
Open access link: scripts.iucr.org/cgi-bin/paper?ge5036
A complete printable manual, installation packages, HOWTO videos, and tutorials with test data can be found on our Source Forge site: sourceforge.net/projects/bioxtasraw.
The manual, tutorial, installation instructions, and links to the videos are also available as a website at: http://bioxtas-raw.readthedocs.io
Thanks to Jesper Nygaard, Kurt Andersen, Alvin Acerbo, and the many BioSAXS users whose feedback has contributed greatly to RAW. Thanks also to SAXSLAB (saxslab.com)
Submitted by: Richard Gillilan and Jesse Hopkins, MacCHESS, Cornell University 09/14/2017
MacCHESS crossed a new threshold in June 2017 with international data collection. Normand Cyr collected data at CHESS beamline F1 remotely from the D'Avanzo lab at the University of Montreal in Quebec, Canada.
Remote data collection, made possible by the use of a sample automounter such as the BAM-2 located at F1 and remote desktop software, is a growing trend at MacCHESS. Research groups regularly ship in their samples from as far away as Texas, and then operate the beamline over an Internet connection to the MacCHESS F1 data collection workstation. Instructions for carrying this out are available on the MacCHESS web site. The D'Avanzo beam time in June is the first time for MacCHESS that the remote operation has crossed international borders.
The D'Avanzo data collection in June was part of another growing trend: combined local/remote data collection. Several members of the group traveled from Montreal to Ithaca, carrying their samples across the border, while Cyr stayed behind. Local collection carries several advantages, including the ability to mount fresh as well as pre-frozen crystals, the ability to interact with MacCHESS staff, and the possibility of trying novel experimental procedures. When his colleagues took time off to sleep, Cyr filled in the beam time with collection of data from his samples over the Internet. Afterwards, Cyr responded, "Thanks for your help during the data collection. On our side, everything went smoothly with the 'hybrid' local/remote data collection."
Submitted by: David J. Schuller, MacCHESS, Cornell University 09/03/2017
The 2017 CHESS Users' Meeting was held on Tuesday and Wednesday, June 6 - 7, 2017 in the Physical Sciences Building located on the Cornell University Ithaca campus. Like most years, the first day of the meeting covered facility-wide improvements and programs, a student paper prize, a poster session, tours and dinner banquet. The second day this year was special, however, hosting two science workshops as well as the annual meeting of the INCREASE organization.
Unlike most years, this year there are unusual external forces shaping the CHESS running schedule and planning for the CHESS future. As highlighted in a previous month’s eNews cover, CHESS Director Joel Brock discussed how CHESS is beginning to plan for another five years of operations starting in 2019, and how CHESS will transition from NSF being the steward to a partnership between NSF, other government agencies, and industry. Brock points out that this presents a rare opportunity to reorganize and do exciting things that we haven't been able to do in the past. He’s started a process of developing a funding model in which partner organizations provide financial support in exchange for both dedicated research capabilities and guaranteed x-ray beam time. We expect that the Users Executive Committee will be involved in the planning process. More news will come in future months.
The first science talk was given by the CHESS student paper prize winner, Mark Weidman (MIT) presented “Kinetics of the self-assembly of nanocrystal superlattices measured by real-time in situ X-ray scattering”. In the afternoon a poster session led to two prizes: Stephen Meisburger (Princeton) won the Best Technical poster with “Unmixing Enzyme Allostery”. Thomas Derrien (Cornell) was awarded the prize of Best Scientific poster with “Dynamics of DNA-Capped Nanoparticle Superlattice Assembly”.
Poster session in atrium of Cornell Physical Sciences Building.
Two science workshops ran in parallel, covering new opportunities in biology and engineering sciences. The biology workshop “Emerging Frontiers in Biology using Synchrotron Radiation” covered two topics: 1) “Advances in Serial Synchrotron Crystallography”, organized by MacCHESS scientist Aaron Finke and 2) “Chemical and Structural Imaging in Biology”, organized by CHESS scientist Arthur Woll.
The structure-focused workshop used as motivation how the advent of high-speed x-ray detectors over the past decade, coupled to advances in the brightness of synchrotron radiation sources, is opening new ways to capture dynamics of proteins and living systems on relevant timescales and with varying degrees of avoiding radiation damage impact on data quality. This workshop discussed in detail the motions of biomolecules (proteins, nucleic acids, complexes) which occur as they perform their functions. “Motion” includes conformational changes, ranging from large domain “hinging” motions to relatively small loop motions (e.g. to allow and deny access to an active site); oligomerization changes; interaction with partner molecules.
The imaging-focused workshop was motivated by the fact that advanced, SR-based x-ray microscopies are finding increasing application in a broad array of fields, including biology, geology, and cultural heritage applications. This growth is driven by many factors – especially the routine availability of micron- and sub-micron-scale x-ray beams, novel detection schemes and detector technology, as well as new and growing user communities in a wide variety of application areas – from biology to cultural heritage. This session brought world leaders in synchrotron science and application areas to explore and identify the most exciting scientific and instrumentation targets for CHESS to pursue in the context of an upgrade. Particular areas of emphasis are: 1) applications in biology, especially plant sciences, 2) opportunities for x-ray microprobe work at high incident energy (30-90 keV), and 3) critical optics and detector technologies required to fully capitalize on these opportunities.
The engineering workshop “Emerging Frontiers in Engineering using Synchrotron Radiation” also covered two topics: 1) “Measuring and Modeling Damage Evolution” and 2) “Quantifying Stress using X-ray diffraction under Multiaxial Loading Conditions”. Both were organized by CHESS scientist Darren Pagan. The first focused on understanding the current state of the field regarding the ability to accurately model the evolution of damage (microscale voids and cracks which lead to material failure). To correctly model damage evolution, experimental data is necessary to calibrate and validate these predictive models. In this session, researchers presented current work modeling the evolution of damage at the microscale, an overview of a wide range of computed tomography capabilities, and discussed possibilities of combining small angle X-ray scattering measurements with X-ray diffraction measurements during in-situ processes.
The second session had presentations from experimentalists who have developed new capabilities to apply multiaxial loads to specimens during in-situ X-ray experiments. Currently, the majority of mechanical testing is performed by applying uniaxial loads to specimens, however, materials in service rarely experience these loading conditions creating disparities between expected and actual behavior. The presenters showed current work studying materials under multiaxial stress states along with a special focus on experimental equipment designed to make these experiments possible.
Also different from past meetings, this year both workshops had awards from the NSF Biology and Engineering divisions to help support the travel of participants. Those funds were well appreciated and impactful.
In addition to the above meeting and two workshops, this year CHESS is hosting a special workshop for the INCREASE organization. INCREASE is the Interdisciplinary Consortium for Research and Educational Access in Science and Engineering, an organization that promotes research and education at minority-serving institutions (MSIs) with a goal to increase the utilization of national research facilities, and for research education and training for members of groups underrepresented in science and engineering research, such as African-Americans, Hispanics, and women (http://www.increaseonline.org/). Supported by an additional award from the NSF Division of Materials Research (DMR), this workshop was integrated into the annual CHESS Users’ Meeting for the expressed reason to facilitate connecting MSI faculty to the CHESS facility users and in-house experts. Participants learned about NSF funding opportunities, particularly the Partnership for Research and Education in Materials (PREM) program. PREM is a program to enhance diversity in materials research and education by stimulating the development of formal, long-term, collaborative materials research and education partnerships between minority-serving institutions and the NSF DMR-supported groups, centers, and facilities.
Overall it was an extremely busy and successful meeting attended by many in the user community. A more complete report of the workshops will be published in the journal Synchrotron Radiation News in the coming months. Interested readers can find the speakers, presentations titles and abstracts still posted at the meeting website.
Submitted by: Ernest Fontes, CHESS, Cornell University 08/11/2017
Cornell Chronicle. By Tom Fleischman
Figure 1: A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo. (a) Schematic of the native E. coli disulfide-bond-formation pathway, which involves the endogenous transmembrane enzyme DsbB. DsbB is located in the inner membrane and interacts with its soluble periplasmic partner DsbA, which is localized to the periplasmic compartment by an N-terminal signal peptide specific for the cotranslational signal recognition particle (SRP) pathway. Electron transport is represented by the black arrows. DsbB obtains its electrons directly from quinones (Q). (b) Expression of DsbB as a soluble biocatalyst in the E. coli cytoplasm is accomplished by using the SIMPLEx technology, which renders IMPs water soluble by introduction of a 'decoy' domain (cMBP) and a 'shield' domain (ApoAI*). DsbA is redirected to the cytoplasm through removal of its native signal peptide. After coexpression, solubilized SxDsbB and export-defective DsbA (cDsbA) effectively transform the cytoplasm into a disulfide-bond-formation compartment. If needed, cDsbA expression can be improved by fusion to E. coli GST, a resident cytoplasmic protein that promotes solubility of its fusion partners.
BioSAXS Essentials 7 participants. Lectures were held atop the new Physical Sciences Building overlooking the Cornell Campus and Cayuga Lake. All but 4 participants were from outside Cornell.
As in past years, we configured both G1 and F1 as essentially identical BioSAXS stations to give more students a chance at hands-on experience during the course. Many students were able to bring their own samples to test for the first time. This year, we were fortunate to have two running size exclusion chromatography (SEC) systems attached directly to the beamlines. These “SEC-SAXS” configurations greatly expand the number of samples that can be successfully analyzed by allowing researchers to separate complex mixtures in real time. MacCHESS is unique in the United States in running two such setups simultaneously for SEC-SAXS. Students were introduced to our RAW software, a very easy to use application for collecting and analyzing SAXS and SEC-SAXS data. RAW is available free for Mac, Windows, and Linux, so students were able to install the program on their laptops to take home (https://sourceforge.net/projects/bioxtasraw/). Steve Meisburger (Ando Group, Princeton) recently published a powerful new algorithm for analyzing difficult-to-separate species in SEC-SAXS, the Evolving Factor Analysis (EFA). Jesse Hopkins (MacCHESS) has created the first publicly available implementation of EFA in our RAW software and students got to learn how to use it on their data. Kushol Gupta (UPENN, Perelman School of Medicine) talked about how he used RAW and EFA to solve a difficult separation problem in his own research.
The course started with Richard Gillilan (MacCHESS) giving the introductory lectures on basic SAXS theory and practice. Since proper sample preparation is essential to success in BioSAXS, Kushol Gupta gave a lecture devoted exclusively to that topic. Thomas Grant (HWI) continued the development of SAXS theory and practice with emphasis on shape reconstruction and other more advanced concepts. The three final lectures of the first day were devoted to advanced methods. Steve Meisburger lectured on SEC-SAXS technique, introducing his EFA method. Kushol Gupta discussed how to deal with mixtures, and described how to use contrast variation to locate polynucleotides within protein complexes. Finally, Thomas Grant showed some preliminary results from a very exciting new method to reconstruct actual electron density from SAXS data.
The Wednesday morning session, led by Jesse Hopkins and Steve Meisburger, was devoted to software tutorials. Students installed our RAW software on their laptops and learned how to process data. In addition to basic quality assessment and common processing practice, they learned how to analyse SEC-SAXS data including the novel EFA method.
The tutorial was followed by two days of intensive around-the-clock data collection by students in small groups. Students were able to choose between regular robotic BioSAXS and SEC-SAXS. Both stations were virtually identical in operation, so students trained on F1 find themselves fully competent to operate G1 when they return for normal research beamtime in the future.
Many thanks to all who helped make this training workshop a success!
BioSAXS Essentials students learn data collection skills from Jesse Hopkins on G1 station (top center). F1 station, normally used for crystallography, was configured as an operationally identical BioSAXS station for the course (Richard Gillilan, bottom left). Both stations supported inline size exclusion chromatography (SEC-SAXS), a popular new technique for separating complex mixtures of biomolecules. During the course, CHESS was the only facility in the nation running two simultaenous SEC-SAXS systems. Also shown: Pedro De La Torre, Michael Durney, and Balasubramnian Harish.
Submitted by: Richard Gillilan, MacCHESS, Cornell University 06/11/2017
Cornell Chronicle. By Elodie Gazave.
Cartoon representation of a 'dolphin-like' single subunit of the apo pdP2X7 structure. Fourteen beta strands are labeled as ß1-14. Each domain is colored consistent with the previous studies for better comparison
Akira Karasawa*, Toshimitsu Kawate*. "Structural basis for subtype-specific inhibition of the P2X7 receptor". eLife, 2016.
*Cornell University, United States