Central challenges in the design of large and dynamic macromolecular assemblies for synthetic biology lie in developing effective methods for testing design strategies and their outcomes, including comprehensive assessments of solution behavior. The authors of this paper created and validated an advanced design of a 600-kDa protein homododecamer that self-assembles into a symmetric tetrahedral cage. The monomeric unit is composed of a trimerizing apex-forming domain genetically linked to an edge-forming dimerizing domain. Enhancing the crystallographic results, high-throughput small-angle x-ray scattering (SAXS) comprehensively contrasted our modifications under diverse solution conditions. To generate a phase diagram associating structure and assembly, we developed force plots that measure dissimilarity among multiple SAXS data sets. These new tools, which provided effective feedback on experimental constructs relative to design, have general applicability in analyzing the solution behavior of heterogeneous nanosystems and have been made available as a web-based application. Specifically, our results probed the influence of solution conditions and symmetry on stability and structural adaptability, identifying the dimeric interface as the weak point in the assembly. Force plots comparing SAXS data sets further reveal more complex and controllable behavior in solution than captured by our crystal structures. These methods for objectively and comprehensively comparing SAXS profiles for systems critically affected by solvent conditions and structural heterogeneity provide an enabling technology for advancing the design and bioengineering of nanoscale biological materials.
Yen-Ting Lai1, Greg L. Hura, Kevin N. Dyer, Henry Y. H. Tang, John A. Tainer and Todd O. Yeates Designing and defining dynamic protein cage nanoassemblies in solution 14 Dec 2016:Vol. 2, no. 12
In this paper, the authors show that CRY1, a protein coding gene that activates circadian gene expression and metabolic states and circadian oscillators, binds directly to the PAS domain core of CLOCK:BMAL1. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24 hr periodicity of gene expression. Integrative modeling and solution X-ray scattering studies (conducted at the SIBYLS beamline 12.3.1) irrefutably position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. This study is significant for understanding the clock mechanism as fundamental for the development and application of therapies for circadian-related disorders.
SAXS profile of CRY1:CLOCK:BMAL1 repressive complex.
(A) Scattering traces of CRY1:CLOCK:BMAL1 ternary complex (CCB) at different con- centrations are shown. These scattering plots were merged to generate the dataset as the input for FoXSDock. (B) Guinier analysis of CCB shows little or no aggregation of sample. SAXS-calculated molecular weight of the ternary complex is 113 kDa. (C) Kratky plot shows the CCB complex indicates a folded mass with an elongated shape. (D) PDB of FoXSDock HADDOCK driven model that is among the top 20 nearly degenerate docking structures, χ = 2.74.
Michael AK, Fribourgh L, Chelliah Y, Sandate C, Hura GL, Schneidman-Duhovny, Tripathi SM, Takahashi JS, Partch CL “Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1” PNAS 2017 Jan 31, doi:10.1073/pnas.1615310114
Apoptosis-inducing factor (AIF) is critical for mitochondrial respiratory complex biogenesis and for mediating necroptotic parthanatos; these functions are seemingly regulated by enigmatic allosteric switching driven by NADH charge-transfer complex (CTC) formation. In this paper the authors define molecular pathways linking AIF’s active site to allosteric switching regions by characterizing dimer-permissive mutants using small-angle X-ray scattering (SAXS) and crystallography and by probing AIF-CTC communication networks using molecular dynamics simulations. Collective results identify two pathways propagating allostery from the CTC active site: (1) active-site H454 links to S480 of AIF’s central β-strand to modulate a hydrophobic border at the dimerization interface, and (2) an interaction network links AIF’s FAD cofactor, central β-strand, and Cβ-clasp whereby R529 reorientation initiates C-loop release during CTC formation. This knowledge of AIF allostery and its flavoswitch mechanism provides a foundation for biologically understanding and biomedically controlling its participation in mitochondrial homeostasis and cell death.
Tip link filaments convey force and gate inner-ear hair-cell transduction channels to mediate perception of sound and head movements. Cadherin-23 and protocadherin-15 form tip links through a calcium-dependent interaction of their extracellular domains made of multiple extracellular cadherin (EC) repeats. These repeats are structurally similar, but not identical in sequence, often featuring linkers with conserved calcium-binding sites that confer mechanical strength to them. In a paper recently published in Nature Communications the Sotomayor lab reports the X-ray crystal structures of human protocadherin-15 EC8-EC10 and mouse EC9-EC10, which show an EC8-9 canonical-like calcium-binding linker, and an EC9-10 calcium-free linker that alters the linear arrangement of EC repeats. Molecular dynamics simulations and small-angle X-ray scattering experiments support this non-linear conformation. Simulations also suggest that unbending of EC9-10 confers some elasticity to otherwise rigid tip links. The new structure provides a first view of protocadherin-15’s non- canonical EC linkers and suggests how they may function in inner-ear mechanotransduction, with implications for other cadherins.
The BioSAXS workshop at the ALS User Meeting on October 4th and 5th was a big success. The workshop was filled to capacity with attendees coming from many different regions of the US. Participants were given detailed information about the range and capabilities of our new Pilatus detector and the future upgrades to the SIBYLS beamline. The latest advances in SAXS studies on biological systems were discussed, along with updates on current developments of software for SAXS analysis. The afternoon of the second day of the workshop was dedicated to practical hands-on SAXS analysis.
We would like to extend special thanks to our illustrious speakers; Haydyn Martens (EMBL,Hamburg), Frank Gabel (IBS, Grenoble), Robert Rambo (Diamond Light Source, Oxford) and John Tainer (M.D. Anderson Cancer Center, Houston) for their informative talks. Thanks also to Subrata Dutta, Henry Tang, Mitra Sayantan, Rahul Banerjee and Ryan Spencer for presenting their SAXS related projects, facilitating some lively, illuminating conversation. And, of course, thanks to everyone who attended.
For those who were not able to attend this year, we look forward to seeing you in October 2017 for the 8th Annual BioSAXS workshop. In the meantime, be sure to take advantage of our Mail-in HTSAXS program and check out all the different SAXS analysis tools that you can download from our website.
We are pleased to announce the 7th annual SIBYLS bioSAXS workshop:
Date: October 4th - 5th, 2016
Location: Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory, Berkeley, CA
The 7th annual SIBYLS bioSAXS workshop will cover frontiers in Biological SAXS. The two-day workshop will provide participants with software tutorial sessions for biological SAXS in addition to hands-on training in experimental techniques. The latest advances in SAXS studies on biological systems will be discussed with particular focus on advances in synchrotron scattering techniques, dynamic and flexible structures in biomolecule, membrane protein scattering, and complementary methods in crystals and in solution. Updates on current developments of software for SAXS analysis pertaining to structural biology will be illustrated.
The first day of the workshop will begin with a brief run-through on current updates. Greg Hura, Berkeley Lab’s SAXS Beamline Scientist at SIBYLS, will introduce the capabilities of the new detector and the future of high throughput SAXS at the SIBYLS Beamline 12.3.1. The keynote speakers, Frank Gabel (IBS, Grenoble), Haydyn Mertens (EMBL, Hamburg), and Robert Rambo (Diamond, Oxford) will continue Dr. Hura’s discussion by elaborating on the basics of SAXS.
Michal Hammel, another SAXS Beamline Scientist at SIBYLS, will give a talk about SAXS modeling, SAXS profile computations using FOX, and calculations of SAXS shape.
Other distinguished speakers, (TBD), will contribute to the basis of the workshop over the two days by sharing complementary experimental approaches and modeling techniques. This will provide for a flux of ideas among workshop participants, and inspire new perspectives for future data analysis. The second day of the workshop will be dedicated to practical hands-on exercises.
Enrollment is limited to 30 participants.
Organizers: Michal Hammel, Greg Hura
Inquires: Kathryn Burnett
Registration: Registration is now open. To attend the workshop you need to REGISTER for the 2016 Advanced Light Source User Meeting. When you register, indicate that you plan to attend the “7th Annual SIBYLS bioSAXS Workshop”.
Tuesday, October 4th (Building 2, Rm 100B)
12:20 Lunch (ALS Patio and Exhibitor Tent)
14:00 Welcoming Remarks: Michal Hammel, LBNL
14:15 Frank Gabel, IBS, Grenoble
15:00 Coffee Break
15:15 Haydyn Mertens, EMBL, Hamburg
16:00 Coffee Break
16:15 John Tainer, The University of Texas M.D. Anderson Cancer Center, Houston
16:30 Update on SAXS at SIBYLS : Greg Hura, LBNL
16:45 Mail-in SAXS at SIBYLS : Kathryn Burnett, LBNL
17:00 End of first day’s workshop
Wednesday, October 5th (Building 2, Rm 100B)
9:00 Short presentations followed by open discussion
10:30 Coffee Break
10:45 Robert Rambo, Diamond Light Source, Oxford
12:00 Lunch (ALS patio and Exhibitors Tent)
13:00 Practical session with mentors (Greg Hura, Michal Hammel, Robert Rambo, Haydyn Martens, Frank Gabel)
16:45 Closing comments, Michal Hammel, LBNL
17:00 End of BioSAXS workshop
The facilities and staff at the SYBILS beamline contributed to this breakthrough study exploring the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain.
Naturally occurring proteins—chains of amino acids that fold into functional, three-dimensional shapes—are believed to represent just a small fraction of the universe of all possible permutations of amino-acid sequences and folds. How can we begin to systematically sift through those permutations to find and engineer from scratch (de novo) proteins with the characteristics desired for medical, environmental, and industrial purposes? To address this question, a team led by researchers from the Institute for Protein Design at the University of Washington have published a landmark study that used both protein crystallography and small-angle x-ray scattering (SAXS) at the ALS to validate the computationally designed structures of novel proteins with repeated motifs. The results show that the protein-folding universe is far larger than realized, opening up a wide array of new possibilities for biomolecular engineering.