Join us in Lund for a lunch-to-lunch scientific symposium from Wednesday (October 9th) to Friday (October 11st), 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 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.
* Please note that if you click on a specific talk in the Timetable, you will come to a page showing the full abstract!
Prof. Yvonne Jones, University of Oxford, UK
Talk - From structure to mechanism in the assembly and modulation of cell surface signalling complexes
Prof. Yifan Cheng, UCSF, USA
Talk - Single particle cryo-EM of membrane proteins
Prof. Nieng Yan, Princeton University, USA
Talk - How is electrical signal generated? Structural and mechanistic investigations of Nav channels
Prof. Mei Hong, MIT, USA
Talk - Structure and Dynamics of Amyloid Proteins from Solid-State NMR: Glucagon & Tau
Prof. Richard Neutze, University of Gothenburg, Sweden
Talk - Time-resolved diffraction experiments at X-ray free electron lasers reveal ultrafast structural changes in photosynthesis
Prof. Erik Lindahl, Stockholm University
Talk - Deciphering Allosteric Modulation in Ligand-Gated Ion Channels with Simulations, X-ray crystallography, Cryo-EM and Neutron Scattering
Dr. Andrey Gruzinov, EMBL Hamburg, Germany
Talk - Small-angle X-ray scattering (SAXS) for integrative structural biology
Prof. Liz Carpenter, University of Oxford, UK
Talk - Using Structural biology of human membrane proteins to understand the causes of genetic diseases
Prof. Poul Nissen, Aarhus University, Denmark
Talk - Structure and Dynamics of Membrane Transport Proteins
Prof. Henning Tidow, Hamburg University, Germany
Talk - Structural studies of integral membrane proteins using stealth carrier nanodiscs
The LINXS Integrative Structural Biology theme is established to promote work that will make it easier for the life science community to use and combine different structural biology methods. Following up the highly successful 1st LINXS Symposium on Integrative Structural Biology that was held in Lund in November 2018, we are now inviting interested to join us for the 2nd LINXS Integrative Structural Biology Symposium that will focus on advanced, cutting-edge research in structural biology. The program is centered around invited internationally leading keynote speakers, that will be mixed with shorter contributed talks selected from the submitted abstracts (expected for PhD-students and postdocs), as well as poster sessions.We especially encourage PhD students and young researchers to attend the meeting to discuss science and benefit from this excellent platform for national and international networking. The target group is researchers from academia as well as researchers from industry and large-scale facilities such as MAX IV and ESS. Both experienced specialists and researchers with more recently established interests in integrative structural biology are expected to greatly benefit from the symposium.
The registration deadline has been extended through September 28.
Using membrane protein crystallography, small-angle scattering techniques, and cryo-EM, and also a range of biochemical and biophysical methods such as electrophysiology, single-molecule FRET, and molecular dynamics simulations, we have obtained deep insight into the functional cycle of primary active transporters of the P-type ATPase family. These transport ATPases are fundamental to physiology, and malfunctions are linked to diseases such as neurological and cardiovascular disorders.
The transmembrane gradients for the key cations Na+, K+, and Ca2+ are generated by Na+,K+-ATPase and Ca2+-ATPases. In brain, Na+,K+-ATPase activity accounts for an estimated 40-70% of total ATP hydrolysis and potentiates e.g. Na+ and K+ channels for their activity in action potentials, membrane potential, and Na+ coupled transport of e.g. glucose, metabolite, neurotransmitters, Ca2+ efflux, pH and Cl- control. Ca2+-ATPases maintain steep calcium gradients, internal Ca2+ stores, and cytoplasmic free calcium at accurate levels that define and potentiate calcium signalling pathways.
Lipid flippases, also of the P-type ATPase family (P4-ATPases) maintain asymmetric lipid distributions in biomembranes. Their activity potentiates membrane dynamics, but the structure and function of lipid flippases remained enigmatic until recently. We determined the first structures of lipid flippases using cryo-EM and revealed at the same time a detailed insight into lipid recognition and autoregulation.
The talk will cover methodological approaches supporting the functional and mechanistic insight we have gained.
The multivesicular sorting machinery is a crucial mechanism for targeting membrane proteins
for recycling or degradation. The lysosomal trafficking regulator-interacting protein 5 (LIP5)
which coordinates the action of this machinery is also known to bind directly to the membrane
protein cargo. In case of aquaporin 2 (AQP2) the binding of LIP5 during the endocytic pathway
in kidney collecting duct cells ensures an effective regulation of urine volume .
In our group, we have previously studied the role of AQP2 phosphorylation in AQP2-LIP5
interaction . Currently we are focusing on elucidating the structural details of the complex
in order to better understand how membrane proteins are delivered to the multivesicular
bodies. We have constructed alanine mutants of single residues in the proposed binding sites
of both AQP2 and LIP5. Studying the binding affinity of these mutants using fluorescence
quenching helps us understand which residues are directly involved in the binding.
Further, AQP2 was successfully incorporated into MSP-based nanodiscs and negative stain
electron microscopy confirmed homogeneous state of the particles. We have collected high
resolution images on Titan Krios and are currently processing the data.
 B. W. M. Van Balkom, M. Boone, G. Hendriks, E. Kamsteeg, J. H. Robben, H. C. Stronks, A.
Van Der Voorde, and F. Van Herp, “LIP5 Interacts with Aquaporin 2 and Facilitates Its
Lysosomal Degradation,” pp. 990–1001, 2009.
 J. V. Roche, S. Survery, S. Kreida, V. Nesverova, H. Ampah-Korsah, M. Gourdon, P. M. T.
Deen, and S. Törnroth-Horsefield, “Phosphorylation of human aquaporin 2 (AQP2)
allosterically controls its interaction with the lysosomal trafficking protein LIP5,” J. Biol.
Chem., vol. 292, no. 35, pp. 14636–14648, 2017.
In my talk I will present our approach for modeling macromolecular
flexibility of large molecular assemblies and how it can be combined with
sparse experimental data obtained with small-angle and cross-linking
Large macromolecular machines, such as proteins and their complexes, are
typically very flexible at physiological conditions, and this flexibility is
important for their structure and function. Computationally, it can be often
approximated with just a few collective coordinates, which can be computed
e.g. using the Normal Mode Analysis (NMA). NMA determines low-frequency
motions at a very low computational cost and these are particularly
interesting to the structural biology community because they are commonly
assumed to give insight into protein function and dynamics .
One of the challenges in the community is the explanation of solution smallangle
scattering profiles. Very recently, we designed a computational scheme
that uses the nonlinear normal modes  as a low-dimensional representation
of the protein motion subspace and optimizes protein structures guided by the
SAXS and SANS profiles [3,4]. For example, in the CASP12 and CASP13
exercises, this scheme obtained best models for some (3 out of 9 in CASP12)
SAXS-assisted targets [5,6]. Overall, the flexible fitting scheme typically allows
a significant improvement of the goodness of fit to experimental profiles in a
very reasonable computational time. The NMA analysis also allows to
automatically split macromolecules into rigid domains, or to be used together
with the cross-linking data, as we demonstrated in the recent CASP13
 Grudinin, S., Laine, E., & Hoffmann, A. (2019). Predicting protein functional
motions: an old recipe with a new twist. bioRxiv, 703652.
 Hoffmann, A. & Grudinin, S. (2017). J. Chem. Theory Comput. 13, 2123 –
2134. For more information https://team.inria.fr/nano-d/software/nolbnormal-
 Grudinin, S. et al. (2017). Acta Cryst. D, D73, 449 – 464. For more
 Tamò, G. E., Abriata, L. A., Fonti, G., & Dal Peraro, M. (2018). Proteins:
Structure, Function, and Bioinformatics, 86, 215-227.
With the technological breakthroughs in the past few years, single particle cryo-electron microscopy (cryo-EM) has enabled rapid progresses in structure determination of integral membrane proteins, particularly ion channels. The pace of structure determination of integral membrane proteins by single particle cryo-EM is unprecedented in structural biology. With such a rapid progress, it is also very critical to interpretation of cryo-EM density maps carefully to ensure that interpretation is data-driven. I will discuss some practical examples to demonstrate the significance of careful interpretations of single particle cryo-EM density maps.
Furthermore, as a prominent example in structural biology of membrane proteins, structural studies of transient receptor potential (TRP) channel superfamily demonstrated nicely how technological breakthroughs impacts scientific discoveries. As an example of our recent studies of TRP channels by single particle cryo-EM, TRPV5 (transient receptor potential vanilloid 5) represents a unique calcium-selective TRP channel essential for calcium homeostasis. Unlike other TRPV channels, TRPV5 and its close homolog, TRPV6, do not exhibit thermosensitivity or ligand-dependent activation but are constitutively open at physiological membrane potentials and modulated by calmodulin (CaM) in a calcium-dependent manner. Structural studies of truncated and full-length TRPV5 in lipid nanodiscs, as well as of a TRPV5 W583A mutant and TRPV5 in complex with CaM provide novel insights to the mechanism of calcium regulation and reveal a flexible stoichiometry of CaM binding to TRPV5.
Galactomannans are hemicelluloses composed of a β-1,4-linked mannose backbone with α-1,6-galactose substitutions.
They are part of our diet as seed storage polysacchardies and food thickeners and are utilised
by several human gut bacteria (1). One such bacteria, Bacteroides ovatus, contains a gene cluster encoding
two glycoside hydrolase family 26 β-mannanases, BoMan26A and BoMan26B (2). BoMan26B generates a
range of product lengths upon mannan hydrolysis, prefers longer substrates and is less restricted by galactose
side-groups than BoMan26A, which mainly generates mannobiose (3,4). The results suggest that BoMan26B
performs the initial attack on galactomannan, generating oligosaccharides that are further hydrolysed by Bo-
Man26A. Crystal structures of these two enzymes reveal the structural basis for their biochemical differences.
BoMan26B, with galactosyl-mannotetraose bound in subsites –5 to –2, has an open and long active site cleft
with W112 in subsite –5 concluded to be involved in mannosyl interaction (4). Moreover, K149 in the –4 subsite
interacted with the galactosyl side-group of the ligand, which may indicate a preference in for substituted
manno-oligosaccharides (4). BoMan26A instead revealed a narrow active site cleft that is restricted in one end
by a loop, explaining its preference for generating shorter products (6).
X-ray free electron lasers (XFEL) have sparked the development of time-resolved serial femtosecond crystallography (TR-SFX), which is a completely new experimental approach to understanding protein structural dynamics. We have used TR-SFX at the LCLS (an XFEL in California) to probe light-driven structural changes from picoseconds to microseconds in a bacterial photosynthetic reaction centre. These integral membrane proteins harvest sunlight in order to transfer electrons from a special pair of bacteriochlorophylls to quinone molecules that are located on the opposite side of an energy transducing biological membrane. Coupled redox reactions balance the charges and this leads to a net effect of two pumped protons per photon absorbed. TR-SFX studies at the LCLS revealed structural changes on the picosecond time-scale near the special pair (which is photo-oxidized by light) and the tightly bound menaquinone (which accepts an electron from the special pair). These structural results provide novel chemical insight into how protein structural dynamics are able to help to stabilize the charge separated state. With the extension of serial crystallography to synchrotron radiation sources, I argue that time-resolved diffraction studies will become more common in the future as new approaches allow new biological systems to be probed.
In my laboratory we combine crystallographic, biophysical, electron and light microscopy based approaches into integrated structural biology analyses to study the assembly and modulation of cell surface signalling complexes involved in development and tissue homeostasis. We aim to generate mechanistic insights, at atomic resolution, which can be tested by functional studies in vitro and in vivo. I will discuss some of the recent results we have generated by applying this approach to the signalling mechanism of the semaphorin-plexin cell guidance system and to the extracellular modulation of signalling by the morphogen Wnt. Published examples of our work on these two systems include the following:
D. Rozbesky, R.A. Robinson, V. Jain, M. Renner, T. Malinauskas, K. Harlos, C. Siebold, and E.Y. Jones. (2019) Diversity of oligomerization in Drosophila semaphorins suggests a mechanism of functional fine-tuning. Nature Commun. 10, 3691.
Y. Kong=, B.J.C. Janssen=, T. Malinauskas, V.R. Vangoor, C.H. Coles, R. Kaufmann, T. Ni, R.J.C. Gilbert, S. Padilla-Parra, R.J. Pasterkamp and E.Y. Jones (2016) ‘Structural basis for plexin activation and regulation.’ Neuron 91, 548-560
S. Kakugawa=, P.F. Langton=, M. Zebisch=, S. Howell, T.-H. Chang, Y. Liu, T. Feizi, G. Bineva, N. O'Reilly, A.P. Snijders, E.Y. Jones and J.-P. Vincent*. (2015) ‘Notum deacylates Wnts to suppress signalling activity.’ Nature 519, 187-192
Multidrug resistance in bacteria, originating from conjugative gene transfer, is an increasingly common problem
in today’s world. The majority of bacteria that causes hospital infections are of gram-positive origin,
but so far very little is known about their conjugation systems. To remedy this, we aim to determine the
molecular structure and function of conjugation complexes belonging to Type IV Secretion Systems (T4SSs)
from gram-positive bacteria. This will lead to a deeper insight into one of the main processes responsible for
horizontal gene transfer events, including the spread of antibiotic resistance genes in bacteria.
We study the proteins involved in forming the T4SS biochemically, structurally and biophysically. Since grampositive
T4SSs are very dissimilar from their gram-negative counterparts, little can be deduced from the few
gram-negative systems so far studied. Furthermore, they occur in a number of pathogens, such as enterococci,
streptococci and staphylococci. Another aspect that makes gram-positive T4SSs interesting is that they are
used to efficiently transfer not only antibiotic resistance, but also virulence factors.
These megadalton sized systems are built up by i) extracellular adhesion proteins, ii) membrane channel proteins
and iii) intracellular DNA processing proteins. Here, I will present our current understanding of the
T4SS originating from the conjugative plasmid pCF10 of Enterococcus faecalis. Our work on this system
combines molecular biology, biochemistry, X-ray crystallography and Electron Microscopy. This has so far
allowed us to determine structures and understand some of the functions of the adhesion proteins as well as
part of the DNA processing proteins, which will highlight both major differences and similarities between the
gram-positive and gram-negative systems.
The human genome project and subsequent sequencing efforts of thousands of patients and healthy individual provides a wealth of associations between variants in genes and diseases. At the SGC our aim is to create a step-change in drug development by providing tools (proteins, structures, assays and bound small molecules), for proteins that are have associations with genetic disease. We focus in particular on proteins that are associated with neuropsychiatry, cancer, rare and metabolic disease, as well as inflammatory conditions. These “Target Enabling Packages” or TEPs, are made freely available to advance our understanding of the biology of disease and to assist in the design of therapeutics. The Carpenter group focuses on integral membrane proteins, including ion channels, solute carriers, ABC transporters and enzymes. Here, I will discuss three examples of genetic hits, PKD2 in kidney disease, TMEM16K in ataxia and DPAGT1 in congenital myasthenia, for which we have obtained structures, and a wealth of additional improvement in our understanding disease biology. These examples of structures of genetic hits illustrate the power of structural biology, as well as the need for extensive additional information, to provide an understanding of disease biology, which is essential for development of therapeutics.
Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the vacuolar/lysosomal membrane before redistribution to other cellular membranes. Using a combination of crystallography, cryo-electronmicroscopy, biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system, NCR1/NPC2, we recently generated a framework for sterol membrane integration (Winkler et al., (2019)). Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration which clarifies the role of NPC proteins in this essential eukaryotic pathway and which rationalizes mutations in patients with Niemann-Pick disease Type C that I will present at the talk.
Winkler et al, Structural insight into eukaryotic sterol transport through Niemann-Pick Type C proteins, Cell (accepted, 2019)
Small-angle X-ray scattering (SAXS) is a powerful method in the studies of solutions of biological macromolecules and nanostructured systems  allowing one to analyze the structure of native particles and complexes and to rapidly assess structural changes in response to variations in external conditions. Dedicated high brilliance synchrotron beamlines and novel data analysis methods  significantly enhanced resolution and reliability of the structural models provided by SAXS. Very important is the ability of SAXS to quantitatively characterize complicated systems and mixtures in native environments and to see the biomolecules in action by rapidly observing responses to changing physical and chemical conditions (e.g. upon pH or temperature changes, ligand binding etc).
Given the limited information content in the scattering data, robust data analysis and modelling methods are of major importance for broad applications of solution SAXS in biology. To reduce the ambiguity of interpretation, SAXS is often combined with other structural methods like crystallography, NMR and electron microscopy, and also with computational, biophysical and biochemical techniques to build hybrid models. In classical applications, SAXS generally yields low resolution quaternary structure but, very importantly, the method can also help to analyze equilibrium mixtures and to visualize flexible portions of the structures, not seen by the high resolution methods.
In the present talk, modern methods for SAXS data analysis will be presented and illustrated by applications to characterize structures and conformational transitions of biological macromolecules in solution. Recent developments including, in particular, in-line chromatography approaches  will be elucidated and perspectives of the synergistic use of SAXS for integrative modeling utilizing complementary methods will be discussed.
The genetic code of viruses, RNA or DNA, are typically protected in an icosahedral capsid, which is primarily
assembled from over a hundred subunits of the same protein in a spontaneous self-assembly process. Similar
highly efficient assembly processes are ubiquitous in biological systems; viral capsids present a unique platform
to exploit for therapeutic advances in the targeted cellular delivery of cargo packaged within the capsid.
Our research aims to provide a more detailed understanding of how this precise viral capsid protein assembly
process occurs from a pool of single building blocks, and additionally the effect and organization of nucleic
acid present during assembly. Here, we present results from small-angle neutron scattering experiments using
contrast variation to reveal the final assembled structural organization of both the protein and nucleic
acid components from recombinant Hepatitis B virus (HBV) capsid protein and a synthetically prepared RNA
containing the capsid protein binding domain. These data revealed that RNA was localized along the inner capsid
surface. Time-resolved small-angle x-ray scattering (SAXS) experiments were also used to determine the
structure during HBV capsid assembly in the presence and absence of RNA. We employed Bayesian statisticsbased
computational methods to extract kinetic parameters of assembly and the overall size and shape of the
dominant structural intermediates from the SAXS data. Additional single-particle cryoEM reconstructions are
provided to assess the effect of RNA on the resulting assembled capsid structure. The combination of timeresolved
scattering data, Bayesian statistics, and cryoEM structural analysis, provides a framework which not
only describes the viral self-assembly process, but can be extended to other hierarchical assemblies in biology.
Protein misfolding into amyloid fibrils is common not only in neurodegenerative diseases but also in pharmaceutical sciences, where many peptide-based drugs have the tendency to fibrillize, thus impeding solution formulation of the drug. Using solid-state NMR spectroscopy, we have investigated the structure and dynamics of two amyloid fibrils, one formed by the peptide hormone glucagon, which is used to treat diabetic hypoglycemia, and the other formed by the microtubule-binding protein tau, which is found in many neurodegenerative diseases. The glucagon fibril structure is unique among all amyloid proteins known to date: the -sheet is antiparallel rather than parallel hydrogen-bonded, contains two coexisting molecular conformations in a single ultrastructural morphology, and has an extraordinary -strand length of 10 nm. The 1.7 Å resolution structure reveals many stabilizing interactions for the fibril, thus suggesting future strategies for designing glucagon analogs that resist fibril formation. Compared to glucagon, the 40 kDa full-length four-repeat tau protein forms a much more complex amyloid fibril, with the majority of the protein being dynamically disordered. Using an extensive set of multidimensional correlation solid-state NMR techniques, we have determined the repeat domains that constitute the -sheet core, and show that this core has a single molecular conformation. This monomorphic nature for an in-vitro tau fibril is fully consistent with the monomorphic nature of brain-derived tau fibrils known to date, suggesting that in vitro fibrillized tau is a good model for studying in vivo tau fibrils. Further, the segments outside the rigid core, which appear as a “fuzzy coat” in electron micrographs, are heterogeneously dynamic. The repeats excluded from the rigid core exhibits partial mobility and -sheet character, while the proline-rich domains undergo large-amplitude anisotropic motions. These results suggest the structure and dynamics of tau in diseases such as progressive supranuclear palsy, and open the path for designing tau inhibitors and imaging agents.
Membrane protein structure determination - Nieng Yan
Neutrons in Structural Biology - Selma Maric
SAXS for biomolecules – Henning Tidow
Computational modelling in Structural Biology - Erik Lindahl
Crystallography as part of the toolkit for integrated structural biology - Yvonne Jones.
Mitochondria contain approximately 1200 different proteins, 99% of which are synthesized on cytosolic ribosomes and need to be delivered into the right destination through the intermembrane space by transport machineries, such as the TIM chaperone. Currently, the mechanistic and structural details of how the TIM chaperone binds to these mitochondrial proteins remain elusive. To gain structural insight into the binding and chaperone mechanisms, we focused on the complex of the TIM9/10 chaperone and the mitochondrial GDP/GTP carrier membrane protein (Ggc1). Such complexes are difficult to study because they consist of a transiently formed, dynamic complex between two folded proteins and a membrane protein that should be solubilized and bound by the chaperone. X-ray crystallography has revealed the core structure of the free chaperone protein, but because of the dynamic nature and large size (~1400 amino acids) of the complex its structural features have remained elusive. Using an integrative approach that combines biochemical assays, NMR spectroscopy and SAXS it was, however, able to obtain detailed but ambiguous information on the structures of the complex. In particular, the experiments showed that the complex consists of two well-structured (TIM9)3/(TIM10)3 hexamers bound to a mostly disordered Ggc1. In this work, we developed a protocol to integrate such heterogeneous experimental data with a coarse-grained molecular model to provide a description of the conformational ensemble of the TIM9/10-Ggc1 complex. In particular, we used a hybrid structure-based model (to describe the intra-molecular interactions within the folded chaperone), an NMR-derived contact potential for chaperone-client interactions and a knowledge-based potential (to describe the inter-molecular interactions between the chaperones and chaperone-client interactions). We used molecular dynamics (MD) simulations to sample the conformational landscape of the complex, and the resulting coarse-grained conformational ensemble was subsequently converted into all-atom resolution and refined using a Bayesian/Maximum Entropy re-weighting approach using the SAXS data. This allows us to generate a weighted ensemble in agreement with experimental measurement. Such integrative structural modeling method is useful to generate a structural ensemble of large and dynamic proteins in a both efficient and reliable way.
Katharina Weinhäupl, Caroline Lindau, Audrey Hessel, Yong Wang, Conny Schütze, Tobias Jores,
Laura Melchionda, Birgit Schönfisch, Hubert Kalbacher, Beate Bersch, Doron Rapaport, Martha
Brennich, Kresten Lindorff-Larsen, Nils Wiedemann and Paul Schanda. Structural Basis of
Membrane Protein Chaperoning Through the Mitochondrial Intermembrane Space. Cell, 175, 1365-
In bacteria, tryptophan synthesis is performed by the enzymes encoded in the trp operon. The product of the
trpC gene, indole-3-glycerol phosphate synthase (IGPS) catalyzes the indole-forming reaction of tryptophan
synthesis. The reaction mechanism includes a decarboxylation step of the substrate 1-(o-carboxyphenylamino)
1-deoxyribulose 5-phosphate (CdRP). The decarboxylation has been assumed to constitute an essential step
of the mechanism since no activity with the decarboxylated variant of the substrate, phenylaminodeoxyribulosephosphate
(PAdRP), was observed in an early study on IGPS from Escherichia coli (Smith and Yanofsky,
In this study, we demonstrate enzyme-catalyzed formation of the native product IGP from decarboxylated
substrate PAdRP using IGPS from Pseudomonas aeruginosa. Moreover, the crystal structure of P. aeruginosa
IGPS in complex with a substrate analogue was solved to 2.1 Å resolution. By structural comparison to E.coli
IGPS (Wilmanns et al., 1992), we provide structure-based hypotheses on the difference in substrate specificity
between the E.coli and P. aeruginosa homologs.
Smith, B. O. H. and Yanofsky, C. (1962) ‘Enzymes Involved in the Biosynthesis of Tryptophan’, Methods Enzymol.,
5, pp. 794–806.
Wilmanns, M. et al. (1992) ‘Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate
isomerase: Indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 Å resolution’, Journal
of Molecular Biology, 223(2), pp. 477–507.
Structural studies of integral membrane proteins (IMPs) are challenging, as many of them are inactive or insoluble in the absence of a lipid environment. We pioneered an approach making use of fractionally deuterium labelled ‘stealth carrier’ nanodiscs that are effectively invisible to low-resolution neutron diffraction and enable structural studies of IMPs in a lipidic native-like solution environment. We show the potential of the method in a joint small-angle neutron scattering (SANS) and X-ray scattering (SAXS) study of the ATP-binding cassette (ABC) transporter protein MsbA solubilized in the stealth nanodiscs. The data allow for a direct observation of the signal from the solubilized protein without contribution from the surrounding lipid nanodisc. Not only the overall shape but also differences between conformational states of MsbA can be reliably detected from the scattering data, demonstrating the sensitivity of the approach and its general applicability to structural studies of IMPs. In a follow-up project, we could also apply this method to investigate the structural basis for the activation of an essential Ca2+-pump by its regulator calmodulin.
This methodology can be applied to other classes of integral membrane proteins and paves the way for low-resolution structure determination of IMPs in solution using both ab initio and rigid body analysis approaches.
The voltage-gated sodium (Nav) channels are responsible for the initiation and propagation of action potentials. Being associated with a variety of channelopathies, they are targeted by multiple pharmaceutical drugs and natural toxins. We determined the crystal structure of a bacterial Nav channel NavRh in a potentially inactivated state a few years ago, which is a homotetramer in primary sequence but exhibits structural asymmetry. Employing the modern methods of cryo-EM, we determined the near atomic resolution structures of a Nav channel from American cockroach (designated NavPaS) and from electric eel (designated EeNav1.4). Most recently, we have determined the cryo-EM structures of the human Nav channels, Nav1.2, Nav1.4, and Nav1.7 in complex with distinct auxiliary subunits and toxins.These structures reveal the folding principle and structural details of the single-chain eukaryotic Nav channels that are distinct from homotetrameric voltage-gated ion channels. Unexpectedly, the two structures were captured in drastically different states. Whereas the structure of NavPaS has a closed pore and the four VSDs in distinct conformations, that of EeNav1.4 and the human channels is open at the intracelluar gate with VSDs exhibiting similar “up”states. The most striking conformational differenc occurs to the III-IV linker, which is essential for fast inactivation. Based on the structural features, we suggest an allosteric blocking mechanism for fast inactivation of Nav channels by the IFM motif. Structural comparison of the conformationally distinct Nav channels provides important insights into the electromechanical coupling mechanism of Nav channels and offers the 3D template to map hundredes of disease mutations.
Micro-crystal electron diffraction (MicroED) has shown in recent years to be a promising method for determining
macromolecular structures (1–5). It enables structural biologists to study proteins from micron-sized
3D crystals that are too small to be studied by conventional X-ray crystallography. Furthermore, MicroED
can be applied to biomolecules of low molecular weight that are beyond what can so far be resolved by single
particle cryo-EM (6,7). However, up to now, all protein structures determined by MicroED had already been
solved previously by X-ray crystallography. Here, we present for the first time an unknown protein structure
– an R2lox metalloenzyme– solved using MicroED (8). MicroED data were collected from plate-like crystals
with an average size of 2 μm × 2 μm × 0.5 μm. By overcoming challenges in sample handling, cryo-EM specimen
preparation, limited data completeness and low signal-to-noise ratio, we are able to solve the structure
by molecular replacement with a search model of less than 36% sequence identity. The resulting electrostatic
scattering potential map at 3.0 Å resolution is of sufficient quality to allow accurate model building and refinement,
providing biologically relevant information on the enzyme. Our results demonstrate MicroED can be
used for solving novel protein structures, using only standard X-ray crystallography software. These findings
illustrate that electron crystallography has the potential to become a widely applicable tool for revealing new
insights into protein structure and function, opening up new opportunities for structural biologists.
1. Shi, D., Nannenga, B. L., Iadanza, M. G. & Gonen, T. eLife 2, (2013).
2. Nannenga, B. L., Shi, D., Leslie, A. G. W. & Gonen, T. Nat. Methods 11, 927–930 (2014).
3. Yonekura, K., Kato, K., Ogasawara, M., Tomita, M. & Toyoshima, C. Proc. Natl. Acad. Sci. 112, 3368–3373
4. Clabbers, M. T. B. et al. Acta Crystallogr. Sect. Struct. Biol. 73, 738–748 (2017).
5. Xu, H. et al. Structure, 26, 667-675 (2018).
6. Khoshouei, M., Radjainia, M., Baumeister, W. & Danev, R. Nat. Commun. 8, 16099 (2017).
7. Henderson, R. Q. Rev. Biophys. 28, 171 (1995).
8. Xu, H. et al. Sci. Adv. 5, eaax4621 (2019).
Urocanate reductase (UrdA) is a bacterial enzyme that was first characterized in
2012 and shown to reduce urocanic acid resulting in a product imidazole propionate
(1). Unlike similar enzymes fumarate reductases, UrdA hasn’t been well investigated.
Besides being an interesting novel enzyme enabling bacteria to grow in anaerobic
conditions with urocanic acid as electron acceptor (1), UrdA was shown to play a
significant role in human gut microbiota, as imidazole propionate levels are
increased in people with type 2 diabetes and it further affects glucose metabolism
Two domain construct of UrdA, consisting of a FAD binding and a mobile domain
were successfully expressed, purified and crystallized. Four X-ray structures were
obtained depicting different states of the enzyme: ADP bound, FAD bound,
substrate/FAD bound and in complex with product/FAD. The data reveals the overall
structural arrangement of the enzyme as well as the substrate binding mode and
The role of UrdA in imidazole propionate production in relation to type 2 diabetes
makes the first structure of the UrdA of particular importance to our understanding of
1. Bogachev, A. V. et al. (2012), Urocanate reductase of Shewanella. Molecular
Microbiology, 86: 1452-1463.
2. Koh A. et al. (2018) Microbially Produced Imidazole Propionate Impairs Insulin
Signaling through mTORC1. Cell 175: 947-961.
Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium
ion reuptake to internal stores. Several of the structural transitions associated with transport have been characterized
by X-ray crystallography, but critical intermediates of the inward-outward switching are missing.
We combined time-resolved X-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD)
simulations for real-time tracking of concerted SERCA reaction-cycle dynamics in the native membrane. The
TR-XSS pre-pulse model differed in the domain arrangement compared to Ca2E1 crystal structures. A 1.5
ms intermediate showed closure of the cytosolic domains typical of Ca2+- and ATP-bound E1 states. A subsequent
transi-ent state with a 13 ms rise-time showed a novel actuator (A) domain arrangement that exposes the
ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing
of so-far elusive domain rearrangements in a native environment.
Ligand-gated ion channels control the electrical excitation of nerve cells, in particular in the post-synaptic membrane in response to chemical signals mediated by neurotransmitters. These receptors exhibit an amazing diversity in detailed structure and function - some human channels have 15-20 slightly genes, and with five subunits this can theoretically lead to almost a million different oligomers. They are further characterised by adopting both closed, open and desensitised states - and in addition to the neurotransmitters causing normal opening they are subject to secondary control - allosteric modulation - by a number of drugs such as alcohols, benzodiazepines, neurosteroids, and anaesthetics that either potentiate or inhibit the agonist response. I will present our work on understanding the molecular mechanisms of these channels by using a broad range of experimental and theoretical methods, and illustrate that while each method has many shortcomings their combination increasingly enable us to capture different timescales, features, interactions and not least dynamics of important membrane proteins. For ligand-gated ion channels in particular, this has enabled us to explain several key mechanisms, including identifying the separate potentiating and inhibitory binding sites, showing how we can reverse the allosteric modulation of specific channels, and propose detailed functional models even from intermediate-resolution structural data.
Jens Lagerstedt, Susanna Törnroth-Horsefield, Thomas Ursby, Trevor Forsyth, Christine Ziegler