Join us in Lund for a lunch-to-lunch workshop from Monday (Nov 19th) to Wednesday (Nov 21st), noon to noon.
Development in the area of structural biology methods combined with new computational possibilities has highlighted the importance of combining different methods in order to maximize the output.
With the rapid progress and improvements in the fields of macromolecular crystallography, Cryo-EM, small angle scattering, electron diffraction and use of XFELs, the new LINXS theme INTEGRATIVE STRUCTURAL BIOLOGY is formed to advance cutting-edge research and to encourage new users to utilize integrative structure biology to address key scientific questions.
Prof. Henry Chapman, Universität Hamburg, Germany
Prof. Christian Betzel, Universität Hamburg, Germany
Dr. Frank Gabel, Institue de Biologie Structurale, Grenoble, France
Prof. Marius Schmidt, University of Wisconsin, USA
Prof. Tamir Gonen, UCLA, USA
Prof. Christine Ziegler, Universität Regensburg, Germany
Prof. Trevor Forsyth, Institut Laue-Langevin, France and Keele University, UK
Dr. Marta Carroni, Science for Life Laboratories, Stockholm, Sweden
Prof. Bernadette Byrne, Imperial College London, UK
Dr. Thomas Ursby, MAX IV, Lund Sweden
Prof. Mikael Akke, Lund University, Sweden
Dr. Marie Skepö, Lund University, Sweden
Dr. Pål Stenmark, Lund University, Sweden
Prof. Jens Preben Morth, Bioengineering, Technical University of Denmark
Dr Karin Lindkvist, Lund University, Sweden
Dr Annette Langkilde, University of Copenhagen, Denmark
Dr Esko Oksanen, ESS, Sweden
Prof. Maria Sunnerhagen, Linköping University, Sweden
Prof. Irmi Sinning, Heidelberg University Biochemistry Center, Germany
Dr. Johanna Höög, Gothenburg University, Sweden
Jens Berndtsson, Stockholm University, Sweden
The first symposium, which is the kick-off event for the new LINXS theme, will focus on advanced, cutting-edge research in the fields of macromolecular crystallography, Cryo-EM, small angle scattering, electron diffraction, mass-spectroscopy, NMR, and use of XFELs etc. The target group is primarily from academia but also from several large-scale facilities (MAX IV, ESS, etc) and industry. The program is centered around invited internationally leading keynote speakers, that will be mixed with shorter invited talks, TechTalks, as well as with presentations by PhD students and young researchers, and will be an excellent platform for national and international networking. Both experienced specialists and researchers with more recently established interests in integrative structural biology are expected to greatly benefit from the symposium.
Small angle neutron scattering (SANS) provides unique insight into biomacromolecular complexes by combining solvent contrast variation (H2O:D2O exchange) with either natural contrast between different classes of biomolecules (proteins, RNA/DNA, lipids/detergents) and/or by applying artificial contrast, i.e. deuteration of specific biomolecules.
Here, I present results from different biological projects where SANS has played a crucial role by providing unique restraints for structural refinement and interpretation, complementary to other techniques (NMR, EM, crystallography).
In a first couple of examples, I will show how distance and shape restraints from SANS have helped to improve the uniqueness of structural models for two multi-protein-RNA complexes, in combination with NMR restraints and building blocks from crystallography [1, 2]. In a second example, the stoichiometry and internal topology of a highly symmetric, hetero-dodecameric aminopeptidase enzyme complex is revealed by SANS, and conclusions on the assembling process can be drawn in combination with EM data . In a third example combining time-resolved (TR) SANS with online fluorescence, the active unfolding of GFP by an unfoldase could be monitored at a time resolution of 30 seconds, as well as the concomitant conformational changes of the unfoldase . As a last example, I will present SANS data from a segmentally deuterated protein .
 Lapinaite et al. (2013) Nature 502(7472), 519-523.
 Hennig et al. (2014) Nature 515(7526), 287-290.
 Appolaire et al. (2014) Acta Cryst. D 70(Pt 11), 2983-2993.
 Ibrahim et al. (2017) Sci. Rep. 7, 40948.
 Sonntag et al. (2017) Angew. Chem. 56(32), 9322-9325.
MAX IV is the first operational 4th generation storage ring, offering synchrotron radiation for many scientific communities. BioMAX, the first macromolecular crystallography (MX) beamline and one of the first beamlines in user operation, is designed to support all kinds of established crystallography methodologies. The experimental station is equipped with an MD3 micro-diffractometer, an Eiger 16M hybrid pixel detector and an ISARA sample changer.
The beamline runs at a high automation level and allows for complete data collections in seconds. We have recently started implementing serial crystallography at BioMAX. The first experiments have been done with a high viscosity jet device and we are working on making fixed-target scanning available.
Recently a second MX beamline has been funded by the Danish Novo Nordisk Foundation. MicroMAX will become a micro-focusing beamline, which will allow investigations of micrometer sized protein crystals at room temperature using serial crystallography. Using a wide bandpass option, MicroMAX can be exploited also for time resolved crystallography down to the microsecond time resolution range. After a four-year construction period, the beamline will be ready for first experiments.
In the last years there has been an explosion of the cryo electron microscopy single particle technique to get high-resolution structures of protein complexes. Cryo-EM has grown from a niche technique into one of the major structural biology methods, especially for large macromolecular complexes or membrane proteins hard to crystallise. What has been defined as the “resolution revolution” has been driven by the development of more stable electron optics, more sensitive direct electron detectors and better and faster software for image processing. Major efforts are also devoted to the inventions of new methods for sample preparation and for automation of both cryo-EM data acquisition and image processing. In fact, getting a nice sample for optimal imaging and processing the data to obtain correct and most informative structures, still requires specialists’ skills.
This lecture will run through examples of cryo-EM structures both from the literature and the Swedish cryo-EM facility; it will show the basic steps of image recording and image processing analysis to obtain one or multiple 3D maps. Some examples of cryo electron tomography (cryo-ET) will be also given to illustrate what can be achieved with the cryo-EM microscopes available to Swedish researchers at SciLifeLab
My laboratory studies the structures of membrane proteins that are important in maintaining homeostasis in the brain. Understanding structure (and hence function) requires scientists to build an atomic resolution map of every atom in the protein of interest, that is, an atomic structural model of the protein of interest captured in various functional states. In 2013 we unveiled the method MicroED, electron diffraction of microscopic crystals, and demonstrated that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). The CryoEM is used in diffraction mode for structural analysis of proteins of interest using vanishingly small crystals. The crystals are often a billion times smaller in volume than what is normally used for other structural biology methods like x-ray crystallography. In this seminar I will describe the basics of this method, from concept to data collection, analysis and structure determination, and illustrate how samples that were previously unattainable can now be studied by MicroED. I will conclude by highlighting how this new method is helping us understand major brain diseases like Parkinson’s disease; helping us discover and design new drugs; shedding new light on chemical synthesis and small molecule chemistry; and showing us unprecedented level of details with sub atomic resolutions.
Dr. Janina Sprenger
Dr. Sergei Grudinin
Dr. Swagatha Ghosh
Dr. Björn Walse
Dr. Yong Wang
Dr. Vladimir Pevala
Prof. Cecilia Emanuelsson
Dr. Erik Hallin
PhD stud Jennifer Virginia Roche
Prof. Maria Sunnerhagen
At modern micro-beam synchrotron (SR) and Free-Electron-Laser (FEL) beamlines micro-sized crystals are preferred and mandatory for serial diffraction data collection. Therefore, new, advanced and reliable methods to prepare, detect and score 3D micro- and nano-sized crystal suspensions, most suitable for X-ray diffraction experiments need to be established in time. Further, a better understanding of the nucleation process is of fundamental importance to grow crystals of desired dimensions.
Experimental data propose that during crystallization biomolecules pass through a nucleation intermediate. However, till now the nucleation process is discussed in theory and experiment differently (1), and till now no clear and unambiguous information is available. In order to obtain more insights about the process and to obtain supporting evidence for the tow-step nucleation mechanism (2) and theory we investigated the nucleation process and early crystallization events for various proteins, applying complementary biophysical methods (3,4,5).
We particular applied in situ dynamic light scattering (6) , small-angle X-ray scattering and transmission electron microscopy experiments. The data we will present strongly support the existence of a two-step mechanism of nucleation. However, the early process is governed by the formation of liquid dense clusters as initial step, followed by the transition to higher order assemblies inside these clusters (7). After crystal nuclei have formed they continue to grow in size. The desired size for SFX experiments is preferably in the upper nanometer or lower micrometer regime. This guides to a strong demand to develop and establish new methods to analyze, score and optimize protein nano- and micro-crystal suspensions for serial crystallography. To support and facilitate this demand we recently designed and constructed a particular microscope based setup, based on detecting second harmonic generation (SHG) signals of crystalline particles in sample suspensions. This method and setup enhances the already available signal sensitivity to such extend that detection of relative small crystals and crystals with higher symmetry, known to produce rather weak signals, is now possible and distinguishing between amorphous and crystalline particles is possible.
Further, the instrument is equipped with additional channels, which are capable to detect the third harmonic generation signal as well, and the signal of three-photon excited UV-fluorescence.
All in parallel provide most and complementary information(s) about the crystalline state of the sample suspension. Details and experimental data will be presented.
Transient Receptor Potential (TRP) ion channels are non-selective cation channels sharing the membrane topology of six transmembrane helices but varying in sensory N- and C-terminal domains. This variation in cytosolic domains is responsible for the wide range of cellular and environmental signals sensed and transduced by TRP channels. The mechanism how these various extracellular and intracellular stimuli can activate TRP channels is one of the emerging questions in the field. The molecular understanding of TRP channels has been boosted tremendously by cryo-EM single-particle analysis. On the other hand, obtaining well-diffracting crystals of the full-length TRP channels proved to be extremely challenging, nevertheless a few X-ray structures are available. NMR and X-ray studies were rather carried out on stable regions within cytosolic domains of TRP channels, which have been structurally investigated without the membrane part. Combining these results obtained from different structural biology methods gave important mechanistic insights, e.g., into gating, ion permeation and selectivity, as well as into the activation of this enigmatic and medically important membrane protein family.
Histatin 5 is a saliva protein that acts as the first line of defence against oral candidiasis caused by Candida Albicans, and it also possesses bactericidal effects. The antimicrobial activity has been ascribed to the high content of basic amino acids. Histatin 5 also participate in the formation of a protective layer on smooth tooth surfaces, and thereby prevent microbial colonization and stabilize mineral-solute interactions. It is established that various transitional metals, such as zinc, nickel, copper, and iron are intrinsically present in the saliva and it is hypothesized that the metal binding abilities of Histatin 5 plays an important role for the candidacidal mechanism. Histatin 5 binds zinc and copper and possesses definitive metal binding motifs for copper and nickel as well as for zinc. In this presentation I will discuss how we use computer simulations on both the atomistic and coarse-grained level in combination with experimental techniques such as scattering and surface adsorption techniques to achieve a molecular understanding of the systems of interest. Furthermore, I will present our latest results regarding Histatin 5 in solution, its interaction with multivalent ions, and the interaction with bilayers corresponding to model cell membranes. The possibility of using Histatin 5-spermidine conjugate as an anti-fungal drug will also be highlighted.
Macromolecular crystallography changed substantially with the advent of the brightest X-ray sources the world has ever seen, the free electron lasers for hard X-rays (XFELs). The immense brilliance and the ultrashort pulses available at these machines triggered the development of serial crystallography, where a very large number of very small crystals are exposed to the X-ray pulses, one by one and in random orientation. We established time-resolved serial femtosecond crystallography (TR-SFX) at the Linac Coherent Light Source1. We followed the trans to cis isomerization of the central p-coumaric acid chromophore in the photoactive yellow protein in real time from 100 femtoseconds to 3 picoseconds2. With X-ray structures determined on the femtosecond time scale we structurally characterize protein structural changes on the excited state energy surface, as well as the isomerization reaction through a conical intersection, for the first time. Catalysis by bio-medically important enzymes is observed on different, slower time scales. The holy grail of time-resolved crystallography is a practical and general method to investigate enzyme catalysis. We developed such an approach called “mix-and-inject serial crystallography” (MISC) to trigger reactions by diffusion of substrate3, 4. We demonstrate that the approach is feasible using the M. tuberculosis β-lactamase (BlaC). BlaC is an enzyme that promotes broad-scale antibiotic resistance by chemically inactivating β-lactam antibiotics. We characterize the structure of the enzyme-substrate complex and that of a reaction intermediate along the catalytic pathway of the BlaC reaction with a third generation antibiotic (Ceftriaxone)5. This talk aims of addressing how MISC may be feasible at new-generation synchrotron X-ray sources.
J. Tenboer, S. Basu, N. Zatsepin, K. Pande, D. Milathianaki, M. Frank, M. Hunter, S. Boutet, G. J. Williams, J. E. Koglin, D. Oberthuer, M. Heymann, C. Kupitz, C. Conrad, J. Coe, S. Roy-Chowdhury, U. Weierstall, D. James, D. Wang, T. Grant, A. Barty, O. Yefanov, J. Scales, C. Gati, C. Seuring, V. Srajer, R. Henning, P. Schwander, R. Fromme, A. Ourmazd, K. Moffat, J. J. Van Thor, J. C. Spence, P. Fromme, H. N. Chapman and M. Schmidt, Science 346 (6214), 1242-1246 (2014).
K. Pande, C. D. M. Hutchison, G. Groenhof, A. Aquila, J. S. Robinson, J. Tenboer, S. Basu, S. Boutet, D. Deponte, M. Liang, T. White, N. Zatsepin, O. Yefanov, D. Morozov, D. Oberthuer, C. Gati, G. Subramanian, D. James, Y. Zhao, J. Koralek, J. Brayshaw, C. Kupitz, C. Conrad, S. Roy-Chowdhury, J. D. Coe, M. Metz, P. Lourdu Xavier, T. D. Grant, J. Koglin, K. G., R. Fromme, V. Srajer, R. Henning, J. H. C. Spence, A. Ourmazd, P. Schwander, U. Weierstall, M. Frank, P. Fromme, A. Barty, H. N. Chapman, K. Moffat, J. J. Van Thor and M. Schmidt, Science 352 (6286), 725-729 (2016).
C. Kupitz, J. L. Olmos, Jr., M. Holl, L. Tremblay, K. Pande, S. Pandey, D. Oberthur, M. Hunter, M. Liang, A. Aquila, J. Tenboer, G. Calvey, A. Katz, Y. Chen, M. O. Wiedorn, J. Knoska, A. Meents, V. Majriani, T. Norwood, I. Poudyal, T. Grant, M. D. Miller, W. Xu, A. Tolstikova, A. Morgan, M. Metz, J. M. Martin-Garcia, J. D. Zook, S. Roy-Chowdhury, J. Coe, N. Nagaratnam, D. Meza, R. Fromme, S. Basu, M. Frank, T. White, A. Barty, S. Bajt, O. Yefanov, H. N. Chapman, N. Zatsepin, G. Nelson, U. Weierstall, J. Spence, P. Schwander, L. Pollack, P. Fromme, A. Ourmazd, G. N. Phillips, Jr. and M. Schmidt, Struct Dyn 4 (4), 044003 (2017).
M. Schmidt, Advances on Condensed Matter Physics (2013), 1-10 (2013).
J. L. Olmos, Jr., S. Pandey, J. M. Martin-Garcia, G. Calvey, A. Katz, J. Knoska, C. Kupitz, M. S. Hunter, M. Liang, D. Oberthuer, O. Yefanov, M. Wiedorn, M. Heyman, M. Holl, K. Pande, A. Barty, M. D. Miller, S. Stern, S. Roy-Chowdhury, J. Coe, N. Nagaratnam, J. Zook, J. Verburgt, T. Norwood, I. Poudyal, D. Xu, J. Koglin, M. H. Seaberg, Y. Zhao, S. Bajt, T. Grant, V. Mariani, G. Nelson, G. Subramanian, E. Bae, R. Fromme, R. Fung, P. Schwander, M. Frank, T. A. White, U. Weierstall, N. Zatsepin, J. Spence, P. Fromme, H. N. Chapman, L. Pollack, L. Tremblay, A. Ourmazd, G. N. Phillips, Jr. and M. Schmidt, BMC Biol 16 (1), 59 (2018).
Spermatozoa swim using their long tail, which is a motile flagellum. Flagella are complex molecular machines that consists of up to 1000 different proteins, which are neatly arranged around a complex microtubule cytoskeleton consisting of nine doublet microtubules surrounding two central singlet microtubules. Movement occurs when motor molecules attached to one doublet microtubule walks on the neighboring microtubule creating a bend on the flagellum. This motion has to be strictly coordinated and regulated to create the flagellum beat, without which no natural human conception can occur.
Doing the first cryo-electron tomography study of human spermatozoa, we found that inside the lumen of microtubules a complex structure spanned over several micrometers in the spermatozoon end piece. This structure forms an interrupted helix and binds to the inside surface of the tubulin heterodimer, the protein that forms microtubules. This has not been seen in other flagella, which suggests that it might be spermatozoa specific. We named it terminal axoneme intra-lumenal spiral, or TAILS for short.
TAILS might be involved in stabilization of the microtubules which otherwise are constantly growing and shrinking, or it might make the end piece more rigid which would yield more force in the flagellar beat. This study was the first time intact human spermatozoa was visualized using cryo-electron tomography. The discovery of this novel structure shows the need to study human flagella directly, to understand the components involved in spermatozoon swimming.
Hydrogens play a key role in many biochemical processes, but generally they are invisible by X-ray crystallography. Neutrons are scattered by the atomic nuclei, which makes neutron crystallography a general method for determining hydrogen positions. The low brilliance of the available neutron sources leads to some experimental challenges: very large crystals are needed and the incoherent scattering from 1H produces high background. The low flux can be mitigated by using polychromatic Laue diffraction, which leads to more complex data processing. Pulsed neutron sources allow resolving the wavelength using the neutron time-of-flight.
The tetanus neurotoxin (TeNT) is a highly potent toxin produced by Clostridium tetani that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal trans- port. The crystal structure of the tetanus toxin reveals a “closed” domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin’s interaction with its polysaccharide receptor. An integrative analysis combining X-ray crystallography, solution scattering and single particle electron cryo-microscopy reveals pH-mediated domain rearrangements that may give TeNT the ability to adapt to the multiple environments encountered during intoxication, and facilitate binding to distinct receptors.
The uric acid/xanthine H+ symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Over a number of years research in my group has focused on first engineering a UapA construct suitable for structural studies and then on obtaining the high-resolution structure. I will describe both our efforts to stabilize the protein and to the recently published crystal structure of a genetically modified version of UapA in complex with xanthine. The structure reveals that UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. The UapA structure and complementary functional analysis strongly indicate that the dimer is key in transport activity. Based on comparison with the structurally related human Anion Exchanger 1, it seems likely that UapA uses an elevator mechanism to transport substrate across the membrane.
During protein synthesis at the ribosome numerous factors act early on the nascent polypeptide chain. These can be grouped into three major classes – chaperones that assist in folding, enzymes that modify the nascent chain and targeting factors that assist in protein localization. As all of them need access to the nascent polypeptide chain, they utilize partially overlapping binding sites at the ribosomal tunnel exit, but their interplay is poorly understood. Our data provide the structural framework for interactions of co-translational factors at the ribosomal tunnel exit. In yeast, the canonical Hsp70 protein Ssb acts together with the ribosome associated complex (RAC), which consists of the inactive Hsp70 protein Ssz and the Hsp40 protein Zuotin. Together, they form a unique chaperone triad at the ribosome. Structure determination of Ssb and RAC together with ribosome binding studies provide detailed insights into the interplay of this chaperone system, which evolved to link translation and protein folding.
Weyer et al. (2017) NSMB 24:144-151.
Zhang et al. (2017) NSMB 24:611-19.
Gumiero et al. (2016) Nat. Comms. 7:13563.
In yeast (Saccharomyces cerevisiae), complex III and complex IV are respiratory chain complexes capable of transferring electrons to oxygen converting it to water. This process results in the creation of a proton gradient over the inner mitochondrial membrane, which drives the ATP synthesis. Complex III and complex IV in yeast form supercomplexes to assist this process. The structure and functional relevance of the yeast supercomplexes is however largely unknown. In this work we solved the structures of the yeast supercomplexes using single particle cryo-electron microscopy at a resolution range of 3.2-3.5 Å. This work reveals the overall architecture of the supercomplexes in yeast and how they differ from similar assemblies previously described in mammals. We show the first near-atomic structure of yeast complex IV and the protein-protein, and lipid-protein interaction implicated in supercomplex formation. Using the structural insight obtained in this study combined with yeast genetics, we hope to generate and characterize respiratory chains mutants that are unable to form supercomplexes, thus providing a greater insight into their overall function.
NMR provides unique, site-specific information on structure, interactions, and dynamics that is often highly complementary to data obtained by other techniques. Using NMR we can study biomolecular phenomena that defy the old school structure–function paradigm, such as transient or fuzzy interactions mediated by flexible protein segments, or interactions involving rare, high-energy conformations that evade structural characterization by other techniques. The ability to characterize conformational and interaction dynamics on a wide range of time scales gives NMR a special role in integrative structural biology. In some cases, very simple NMR experiments can provide information that is critically important for model building in conjunction with data from other techniques.
Aquaglyceroporins are integral membrane proteins known to facilitate transport of glycerol. The aquaglyceroporin AQP7 is expressed in adipocytes where it regulates glycerol efflux as it translocate to the plasma membrane during lipolysis as a result of catecholamine stimulation. Deletion of AQP7 in mice leads to development of obesity and adipocyte hypertrophy, suggesting an important role in human metabolism. We propose a molecular mechanism where the AQP7 mobility in adipocytes is dependent on perilipin 1 and protein kinase A. Structural analyses combined with ex vivo studies in human primary adipocytes, demonstrate that perilipin 1 binds to AQP7, and that catecholamine activated protein kinase A phosphorylates the N-terminus of AQP7, thereby reducing complex formation.
Three classes of Magnesium transporters have been identified in Bacteria; CorA, MgtE and MgtA/MgtB . While CorA and MgtE are both magnesium channels. Active influx is believed to mediated by MgtA The magnesium transporter A (MgtA) is a specialized P-type ATPase, that import Mg(II) into the cytoplasm. In both Salmonella typhimurium and Escherichia coli. This study demonstrates, for the first time, that MgtA is highly depended on anionic phospholipids and in particular, cardiolipin, the in vitro kinetic experiments performed on detergent solubilized MgtA suggest that cardiolipin act as a magnesium chaperone. We further show that MgtA is highly sensitive to free Mg(II) (Mg(II) free) levels in the solution. MgtA is activated when the Mg(II)free concentration is reduced below 10 uM and is strongly inhibited above 1 mM, indicating that Mg(II) free acts as product inhibitor. Colocalization studies confirm that MgtA is found in the cardiolipin lipid rafts in the membrane. Combined, our findings indicate that MgtA may act as a sensor as well as a transporter of Mg(II) . With the present functional data, we now hypothesize that regulation of ion transport in the MgtA might be fundamentally equivalent to that of the Na+/K+ -ATPase. The discovery that MgtA acts as a receptor in addition to being an ion transporter, is a major breakthrough.