iNEXT-Discovery: Scientific publications


All publications from iNEXT-Discovery acknowledge the project and are appearing as Open Access!



May 14, 2021

Sample preparation strategies for efficientcorrelation of 3D SIM and soft X-raytomography data at cryogenic temperatures

doi: 10.1038/s41596-021-00522-4

3D correlative microscopy methods have revolutionized biomedical research, by providing multidimensional information for in-depth understanding of biological systems. With novel cryo-preservation methods, correlative imaging of cryogenically preserved samples led to nanometer resolution imaging (2–50 nm) under harsh imaging regimes, such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present sample preparation strategies and direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK; BL09 Mistral at ALBA in Spain) and led to the development of a decision aid to facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used.

Okolo CH, Kounatidis I, Groen J, Nahas KL, Balint S, Fish TM Koronfel MA, Cortajarena AL, Dobbie IM, Pereiro E, Harkiolaki M.

Nat Protoc. 2021

 February 12, 2021

Modelling covalent linkages in CCP4

doi: 10.1107/S2059798321001753

Current protocols for modelling covalent linkages within the CCP4 suite are considered. The mechanism used for modelling covalent linkages is reviewed. Previously, linkage descriptions were lacking in quality compared with those of contemporary component dictionaries. Consequently, AceDRG has been adapted for the generation of link dictionaries of the same quality as for individual components. The approach adopted is outlined, which includes associated modifications to the linked components. Several tools to facilitate the practical modelling of covalent linkages are described. The integrated solutions streamline and ease the covalent-linkage modelling workflow, seamlessly transferring relevant information between programs. Recommended practice is illustrated by means of instructive practical examples. By summarizing the different approaches to modelling linkages that are available within the CCP4 suite, limitations and potential pitfalls that may be encountered are highlighted in order to raise awareness, with the intention of improving the quality of future modelled covalent linkages in macromolecular complexes.

Nicholls RA, Joosten RP, Long F, Wojdyr M, Lebedev A, Krissinel E, Catapano L, Fischer M, Emsley P, Murshudov GN

April 13, 2021

The missing link: covalent linkages in structural

doi: 10.1107/S2059798321003934

Here we assesses the extent of problems involving covalent linkages in the Protein Data Bank (PDB). Not only will this facilitate remediation of existing models, but it will also inform and improve the quality of future linkages. By considering linkages of known type in the CCP4 Monomer Library, failure to model a covalent linkage is identified to result in inaccurate (longer) interatomic distances. Scanning the PDB for atom pairs that do not have a corresponding type in the CCP4-ML reveals many commonly occurring types of unannotated potential linkages that may or may not be covalently linked. Manual consideration of the most commonly occurring cases identifies a number of genuine classes of covalent linkages. The need for a universal standard for maintaining link records corresponding to covalent linkages is emphasized. The importance of correctly modelling covalent linkages is demonstrated in a case study that involves the covalent linkage of an inhibitor to the main protease in various viral species, including SARS-CoV-2.

Nicholls RA, Wojdyr M, Joosten RP, Catapano L, Long F, Fischer M, Emsley P, Murshudov GN.

May 10, 2021

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

doi: 10.3389/fmolb.2021.653148

The highly infectious COVID-19 caused by SARS-CoV-2 poses a threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide new approaches by gathering scientific expertise worldwide. Making available viral proteins and RNAs will pave the way to understand the SARSCoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the molecular components provide the essential basis for further work, including macromolecular interaction studies and high throughput drug screening. Here, we present the holistic SARSCoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

Altincekic N, Korn SM, Qureshi NS, Dujardin M, Ninot-Pedrosa M, Abele R, Abi Saad MJ, Alfano C, Almeida FCL, Alshamleh I, de Amorim GC, Anderson TK, Anobom CD, Anorma C, Bains JK, Bax A, Blackledge M, Blechar J, Böckmann A, Brigandat L, Bula A, Bütikofer M, Camacho-Zarco AR, Carlomagno T, Caruso IP, Ceylan B, Chaikuad A, Chu F, Cole L, Crosby MG, de Jesus V, Dhamotharan K, Felli IC, Ferner J, Fleischmann Y, Fogeron M-L, Fourkiotis NK, Fuks C, Fürtig B, Gallo A, Gande SL, Gerez JA, Ghosh D, Gomes-Neto F, Gorbatyuk O, Guseva S, Hacker C, Häfner S, Hao B, Hargittay B, Henzler-Wildman K, Hoch JC, Hohmann KF, Hutchison MT, Jaudzems K, Jovic K, Kaderli J, Kalnins G, Kanepe I, Kirchdoerfer RN, Kirkpatrick J, Knapp S, Krishnathas R, Kutz F, zur Lage S, Lambertz R, Lang A, Laurents D, Lecoq L, Linhard V, Löhr F, Malki A, Bessa LM, Martin RW, Matzel T, Maurin D, McNutt SW, Mebus-Antunes NC, Meier BH, Meiser N, Mompeán M, Monaca E, Montserret R, Mariño Perez L, Moser C, Muhle-Goll C, Neves-Martins TC, Ni X, Norton-Baker B, Pierattelli R, Pontoriero L, Pustovalova Y, Ohlenschläger O, Orts J, Da Poian AT, Pyper DJ, Richter C, Riek R, Rienstra CM, Robertson A, Pinheiro AS, Sabbatella R, Salvi N, Saxena K, Schulte L, Schiavina M, Schwalbe H, Silber M, Almeida MdS, Sprague-Piercy MA, Spyroulias GA, Sreeramulu S, Tants J-N, Tars K, Torres F, Töws S, Treviño MÁ, Trucks S, Tsika AC, Varga K, Wang Y, Weber ME, Weigand JE, Wiedemann C, Wirmer-Bartoschek J, Wirtz Martin MA, Zehnder J, Hengesbach M, Schlundt A.

June 18, 2021

Protocol for image registration of correlativ soft X-ray tomography and super-resolution structured illumination microscopy images

doi: 10.1016/j.xpro.2021.100529

Advances in imaging technology constantly enable breakthroughs in biological sub-cellular research. Cryo-imaging allows for nanometer resolution at near-physiological conditions, and correlative cryo-imaging integrates further information about cellular processes. A recent development in correlative cryo-imaging involves a platform developed at the correlative cryo-imaging beamline B24 at the UK synchrotron facility which combines 3D super-resolution fluorescence microscopy (cryo-SIM) and soft X-ray tomography (cryo-SXT). A crucial step in the analysis of imaging data generated across microscopes within an imaging platform is the accurate image co-registration without systematic positional errors or data distortion in 3D. Here, we demonstrate the use of the open source Icy plugin eC-CLEM for the registration of light microscopy and X-ray tomography volumes in an easy to follow step-by-step process.

Vyas N, Kunne S, Fish TM, Dobbie IM, Harkiolaki M, Paul-Gilloteaux P.

April 8, 2021

FragMAXapp: crystallographic fragment-screening data-analysis and project-management system

doi: 10.1107/S2059798321003818

Crystallographic fragment screening (CFS) is now a major techniques for screening compounds in early stages of drug-discovery projects. Following advances in automation and throughput at macromolecular crystallography beamlines, the bottleneck for CFS has shifted from collecting data to handling the analysis of such projects. The complexity emerging from the use of multiple methods for processing and refinement and to search for ligands requires an equally sophisticated solution to summarize the output, allowing researchers to focus on scientific questions rather than software technicalities. FragMAXapp is a new fragment screening project-management tool designed to handle CFS projects at MAX IV Laboratory. It benefits from powerful computing infrastructure of largescale facilities and, as a web application, it is accessible from everywhere.

Lima GMA, Jagudin E, Talibov VO, Benz LS, Marullo C, Barthel T, Wollenhaupt J, Weiss M, Mueller U.

March 30, 2021 

3D Heteronuclear Magnetization Transfers for the Establishment of Secondary Structures in SARS-CoV-2-Derived RNAs

doi: 10.1021/jacs.1c01914
A heteronuclear-resolved version of NOESY is presented, in which magnetization that transfers between the aqueous solvent and the nucleic acid protons is controlled by selecting specific chemical shift combinations of a coupled 1H-15N spin pair. This leads to a pseudo-3D HSQC-NOESY spectrum with cross-peaks enhanced ~2-5 times compared with conventional 2D NOESY. The enhanced  sensitivity as well as access to both 15N-1H and 1H-1H NOESY dimensions can greatly facilitate RNA assignments and secondary structure determinations.
Kim J, Novakovic M, Jayanthi S, Lupulescu A, Kupce E, Grün JT, Mertinkus K, Oxenfarth A, Richter C, Schnieders R, Wirmer-Bartoschek J, Schwalbe H, Frydman L.
March 26, 2021 

1H, 13C, and 15N backbone chemical‑shift assignments of SARS‑CoV‑2 non‑structural protein 1 (leader protein)

doi: 10.1007/s12104-021-10019-6
The near complete backbone chemical shift assignment of full-length SARS-CoV-2 Nsp1 is presented, revealing its domain organization, secondary structure and backbone dynamics. The results will be of value to further MNR-based investigations of the biochemical and physiological functions of Nsp1.
Ying Wang Y, Kirkpatrick J, zur Lage S, Korn SM, Neißner K, Schwalbe H, Schlundt A, Carlomagno T.
March 21, 2021

Di-phosphorylated BAF shows altered structural dynamics and binding to DNA, but interacts with its nuclear envelope partners

doi: 10.1093/nar/gkab184

It is shown that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are very similar. In solution, however, the flexibility of the N-terminal helix-1 and loop from helix-1 to -2 in BAF is strongly reduced in di-phosphorylated BAF, due to interactions between phosphorylated residues and the positively charged C-terminal helix-6. These regions are involved in DNA and lamin A/C binding. Consistently, phosphorylation causes a huge loss of affinity for dsDNA, but does not impair binding to lamin A/C Ig-fold domain.

Marcelot A, Petitalot A, Ropars V, Le Du M-H, Samson C, Dubois S, Hoffmann G, Miron S, Cuniasse P, Marquez JA, Thai R, Theillet F-X, Zinn-Justin S.

March 9, 2021

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor

doi: 10.3791/62323

We recently reported an NMR bioreactor to maintain a high number of human cells metabolically active for up to 72 h. This setup was applied to monitor protein interactions and chemical modification. We also introduced a workflow for quantitative analysis of the real-time NMR data. The method provides concentration profiles of the chemical species in the cells as a function of time, which can be used to obtain kinetic parameters. Here we provide a detailed description of the NMR bioreactor setup and its application.

Barbieri L, Luchinat E.

May 6, 2021

Cryo-focused ion beam lamella preparation protocol for in situ Structural Biology

doi: 10.1007/978-1-0716-1406-8
The advances in cryo-EM enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since cryo-ET is generally limited to specimens with thickness <500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells was developed and optimized last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from vitrified cells. Preparation of lamellae from large adherent mammalian cells, a small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life-sciences.
Moravcová J, Dopitová R, Pinkas M, Nováček J.
March 8, 2021

Magnetization transfer to enhance NOE cross‐peaks among labile protons: Applications to imino‐imino sequential walks in SARS‐CoV‐2‐derived RNAs

doi: 10.1002/anie.202015948
The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing imino–imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.
Novakovic M, Kupce E, Scherf T, Oxenfarth A, Schnieders R, Grün T, Wirmer-Bartoschek J, Richter C, Schwalbe H, Frydman L.
March 2, 2021

Mixed-valence compounds as polarizing agents for Overhauser dynamic nuclear polarization in solids
In this paper a novel set of polarizing agents is tested by electron paramagnetic resonance methods and their performance si demonstrated in high-field Dynamic Nuclear Polarization (DNP) experiments in the solid state. The mixed-valence compounds used constitute a group of molecules, in which molecular mobility persists even in solids. Consequently, such polarizing agents can be used for Overhauser-DNP experiments in the solid-state, with favorable conditions for DNP at ultra-high magnetic fields.
Gurinov A, Sieland B, Kuzhelev A, Elgabarty H, Kuhne TD, Prisner T, Paradies J, Baldus M, Ivanov KL, Pylaeva S.
Jan 25, 2021

Protein in-cell NMR spectroscopy at 1.2 GHz

doi: 10.1007/s10858-021-00358-w
Here we show the first in-cell NMR spectra recorded at 1.2 GHz on human cells! We compare resolution and sensitivity against those obtained at 900 and 950 MHz. To evaluate the effects of different spin relaxation rates, SOFAST-HMQC and BEST-TROSY spectra were recorded on intracellular α-synuclein and carbonic anhydrase. Major improvements are observed at 1.2 GHz when analyzing unfolded proteins, such as α-synuclein, while the TROSY scheme improves the resolution for both globular and unfolded proteins.
Luchinat E, Barbieri, L, Cremonini M, Banci L.
February 10, 2021

Real-time NMR spectroscopy in the study of biomolecular kinetics and dynamics

doi: 10.5194/mr-2021-16
The review describes the application of NMR spectroscopy to study kinetics of folding, refolding and aggregation of proteins, RNA and DNA. In particular irreversible folding experiments pose large requirements on (i) the signal-to-noise due to the time limitations and (ii) on synchronizing the refolding steps. Here, we discuss the application of methods for signal-to-noise increases including DNP, hyperpolarization and photo-CIDNP for time-resolved NMR. Methods are reviewed ranging from pressure- and temperature-jump, light induction and rapid mixing to induce rapidly non-equilibrium conditions required to initiate folding.
Pintér G, Hohmann KF, Grün JT, Wirmer-Bartoschek J, Glaubitz C, Fürtig B, Schwalbe H.
April 6, 2021

The folding landscapes of human telomeric RNA and DNA G-quadruplexes are markedly different

doi: 10.1002/anie.202100280
We investigated folding kinetics of G-quadruplex (G4) structures. In particular, we compare K+-induced folding of an RNA G4 derived from human telomeric repeat-containing RNA (TERRA25) with a homologous DNA G4 (wtTel25) using CD and real-time NMR spectroscopy. The folding kinetics of these G4's are markedly different: while DNA G4 folding is biphasic, reveals kinetic partitioning and involves kinetically favored off-pathway intermediates, RNA G4 folding is faster and monophasic. The differences in kinetics are correlated to the differences in the folded conformations of RNA vs DNA G4s. Modified DNA G4s with 19F bound to C2’ in arabino configuration adopt exclusively anti conformations for chi. These fluoro-modified DNA (antiTel25) reveal faster folding kinetics and monomorphic conformations similar to RNA G4s, suggesting correlation between folding kinetics and pathways with differences in chi-angle preferences in DNA and RNA, respectively.
Müller D, Bessi I, Richter C, Schwalbe H.
December 20, 2020

Facilitated crystal handling using a simple device for evaporation reduction in microtiter plates

doi: 10.1107/S1600576720016477
In the past decades, most steps in a macromolecular crystallography experiment have undergone tremendous development with respect to speed, feasibility and increased throughput. The part of the workflow that is still a bottleneck is the manipulation and harvesting of the crystals for the diffraction experiment. Here, a novel low-cost device is presented that functions as a cover for 96-well crystallization plates. It enables access to individual experiments one at a time by its movable parts, while minimizing evaporation of all other experiments on the plate. In tests, drops of many crystallization cocktails could be successfully protected for up to 6h. Thus, the manipulation and harvesting of crystals is straightforward for the experimenter. This enables significantly higher throughput which is useful for many macromolecular crystallography experiments, such as screening campaigns.
Barthel T, Huschmann FU, Wallacher D, Feiler CG, Klebe G, Weiss MS, Wollenhaupt J.
Oct 23, 2020

Structural rearrangement of amyloid-beta upon inhibitor binding suppresses formation of Alzheimer's disease related oligomers

doi: 10.7554/eLife.59306
Here, it is shown for two fibril inhibiting ligands - an ionic molecular tweezer and a hydrophobic peptide - that despite their different interaction mechanisms, the suppression of the fibril pathway can be deduced from the disappearance of the corresponding structure of the first amyloid-b oligomers.
Lieblein T, Zangl R, Martin J, Hoffmann J, Hutchison MJ, Stark T, Stirnal E, Schrader T, Schwalbe H, Morgner N.
Dec 14, 2020

Solution structure of the voltage-gated Tim23 channel in complex with a mitochondrial presequence peptide

doi: 10.1038/s41422-020-00452-y
Most mitochondrial proteins are synthesized in the cytosol and transported into mitochondrial sub-compartments mediated by intricate multimeric machineries. Tim23, the key component of the TIM23 complex, forms a channel in the mitochondrial inner membrane (MIM) and is believed to recognize and translocate precursor proteins into the mitochondrial matrix or to release them into the MIM. Previously it was shown that Tim23 forms a protein-conducting channel that can be activated by an appropriate change in the membrane potential in the presence of a mimetic mitochondrial preprotein, a signaling peptide corresponding to the presequence of cytochrome c oxidase subunit IV. Here, we reveal the 3D structure of the yeast Tim23 channel in complex with the presequence in micelles by solution NMR.
Zhou S, Ruan M, Li Y, Yang J, Bai S, Richter C, Schwalbe H, Xie C, Shen B, Wang J.
Dec 10, 2020

PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins

doi: 10.1093/nar/gkaa1021
The Protein Ensemble Database (PED) holds structural ensembles of intrinsically disordered proteins. The new version, PED 4.0, has been redesigned and reimplemented with cutting-edge technology. It holds about six times more data and a broader representation of state-of-the-art ensemble generation methods than before, has a completely renewed graphical interface, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. A new submission process combines both automatic and manual evaluation steps, and a team of biocurators integrate structured metadata. A new search engine allows the user to build advanced queries and search all entry fields.
Lazar T, Martínez-Pérez E, Quaglia F, Hatos A, Chemes LB, Iserte JA, Méndez NA, Garrone NA, Saldaño TE, Marchetti J, Rueda AJV, Bernadó P, Blackledge M, Cordeiro TN, Fagerberg E, Forman-Kay JD, Fornasari MS, Gibson TJ, Gomes GW, Gradinaru CC, Head-Gordon T, Jensen MR, Lemke EA, Longhi S, Marino-Buslje C, Minervini G, Mittag T, Monzon AM, Pappu RV, Parisi G, Ricard-Blum S, Ruff KM, Salladini E, Skepö M, Svergun D, Vallet SD, Varadi M, Tompa P, Tosatto SCE, Piovesan D
Oct 27, 2020

Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

doi: 10.3390/cryst10110975
We describe the updated size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) set-up used at the P12 bioSAXS beam line of the European Molecular Biology Laboratory (EMBL) at the PETRAIII synchrotron, DESY Hamburg (Germany). SEC-SAXS has become a well-established approach to reduce effects of sample heterogeneity on SAXS measurements. The additional use of multi-angle laser light scattering (MALLS), UV absorption spectroscopy, refractive index (RI), and quasi-elastic light scattering (QELS) in parallel to the SAXS measurements enables independent molecular weight validation and hydrodynamic radius estimates. The benefits of the current SEC-SAXS set-up are demonstrated on a set of selected standard proteins.
Graewert MA, De Vela S, Gräwert TW, Molodenskiy DS, Blanchet CE, Svergun DI, Jeffries CM
Oct 16, 2020

Intracellular Binding/Unbinding Kinetics of Approved Drugs to Carbonic Anhydrase II Observed by in-Cell NMR

doi: 10.1021/acschembio.0c00590
In-cell NMR was applied to investigate binding of approved drugs to the isoform II of carbonic anhydrase (CA) in living human cells. We observed strikingly dose- and time-dependent binding, and some drugs exhibited a more complex behavior than others. Some compounds were shown to gradually unbind from intracellular CA II, even in the presence of free compound in the external medium, thus preventing quantitative formation of a stable protein-ligand complex. This could be correlated to the off-target binding activity of these compounds, suggesting that this approach could provide information on the pharmacokinetic profiles of lead candidates at the early stages of multitarget drug design.
Luchinat E, Barbieri L, Cremonini M, Nocentini, A, Supuran CT, Banci L
July 21, 2020

Real-time Quantitative In-Cell NMR: Ligand Binding and Protein Oxidation Monitored in Human Cells Using Multivariate Curve Resolution

doi: 10.1021/acs.analchem.0c01677
Here, an improved bioreactor design is reported, which keeps human cells alive and metabolic for up to 72 h, as well as a novel workflow for quantitative analysis of real-time in-cell NMR data. We monitor protein-ligand interactions and protein oxidation in real time. High-quality concentration profiles can be obtained from noisy 1D and 2D NMR data with high temporal resolution, allowing fitting with kinetic models. The approach can be applied to study complex kinetic behavior of macromolecules in a cellular setting, and could be extended to any real-time NMR application in live cells.
Luchinat E, Barbieri L, Campbell TF, Banci L
Sept 25, 2020

In-cell NMR of functional riboswitch aptamers in eukaryotic cells

doi: 10.1002/anie.202007184
We show the in-cell NMR-spectroscopic observation of the cognate ligand 2’-deoxyguanosine binding to the aptamer domain of the bacterial 2’-deoxyguanosine-sensing riboswitch in eukaryotic Xenopus laevis oocytes and in human HeLa cells. The binding mode established by in vitro characterization of this prokaryotic riboswitch appears maintained in eukaryotic cellular environment. Thus far, in-cell NMR studies on RNA in mammalian cells were limited to short (<15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation. Now we show that in-cell NMR can be used for characterization of much larger (~70 nt) functional and chemically non-modified RNA.
Broft P, Dzatko S, Krafcikova M, Wacker A, Hansel-Hertsch R, Trantirek L, Schwalbe, H
Aug 14, 2020

19-F NMR-based fragment screening for 14 different biologically active RNAs and 10 DNA and protein counter-screens

doi: 10.1002/cbic.202000476
Here the NMR 19F screening of RNA targets with different secondary and tertiary structure is shown to systematically assess the druggability of RNAs. Our RNAs include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. We show that RNA can be specifically targeted and to demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we show how to increase binding affinity from an initial hit using chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low micromolar binding affinity without losing binding specificity between two different terminator structures.
Binas O, de Jesus V, Landgraf T, Völklein AE, Martins J, Hymon D, Berg H, Bains JK, Biedenbänder T, Fürtig B, Gande SL, Niesteruk A, Oxenfarth A, Qureshi NS, Schamber T, Schnieders R, Tröster A, Wacker A, Wirmer-Bartoschek J, Martin MAW, Stirnal E, Azzaoui K, Blommers MJJ, Richter C, Sreeramulu S, Schwalbe H
July 22, 2020

Anomeric Selectivity of Trehalose Transferase with Rare L-Sugars

doi: 10.1021/acscatal.0c02117
The anomeric selectivity of trehalose transferase from Thermoproteus uzoniensis was investigated for both D- and L-glycopyranose acceptors. The enzyme couples carbohydrates to yield trehalose analogues with conversion and enantioselectivity of >98%. The anomeric selectivity inverts from α,α-(1 → 1)-glycosidic bonds for D-glycopyranose acceptors to α,β-(1 → 1)-glycosidic bonds for L-glycopyranose acceptors, while (S)-selectivity was retained for both types of sugar acceptors. Comparison of crystal structures of trehalose transferase in complex with α,α-trehalose and an unnatural α,β-trehalose analogue highlighted the mechanistic rationale for the observed inversion of anomeric selectivity.
Mestrom L, Marsden SR, Van der Eijk H, Laustsen JU, Jeffries CM, Svergun DI, Hagedoorn PL, Bento, I, Hanefeld, U
July 17, 2020

Multitarget Virtual Screening for Drug Repurposing in COVID19

doi: 10.26434/chemrxiv.12652997.v2 (ChemRxiv)
Therapeutic or preventive research for SARS-CoV2 is an extremely active topic of research. Here, we report a virtual drug screening analysis that, to the best of our knowledge, is the widest to date in terms of target proteins and compound library used. We focus on the repurposing of currently already commercialized drugs, especially those shown to interact with multiple viral proteins and having multiple binding sites within each protein. In a second virtual screening analysis we compare our results to predicted binding affinities for the drugs currently in clinical trials. The best molecules in our screening compare favorably to those in clinical trials, suggesting their suitability for therapeutic or preventive applications.
Sorzano CO, Crisman E, Carazo JM, Leon R
July 1, 2020

The basics of small-angle neutron scattering (SANS for new users of structural biology)

doi: 10.1051/epjconf/202023603001
Small-angle neutron scattering (SANS) probes the time-preserved structural state(s) of bio-macromolecules in solution. It allows to assess the redistribution of mass, i.e. changes in conformation, that occurs when macromolecules interact to form higher-order assemblies and to evaluate the structure and disposition of components in such systems. SANS allows ‘out of the box thinking’, from investigating the structures of macromolecules and their complexes to where structural biology interfaces with soft-matter and nanotechnology. All of this simply rests on the way neutrons interact and scatter from atoms (largely hydrogens) and how this interaction differs from the scattering of neutrons from the nuclei of other ‘biological isotopes’.
Jeffries CM, Pietras Z, Svergun DI
May 24, 2020

Improvements on marker-free images alignment for electron tomography

doi: 10.1101/2020.05.22.110445 (BioRXiv)
The Electron Tomography technique to obtain 3D structural information of samples is limited by shifts occurring during acquisition that need to be corrected before the reconstruction process. Earlier, we proposed an approach for marker-free post-acquisition alignment of tilt series images. We improved the method to make it more reliable, stable and accurate and modified the image formation model underlying the alignment procedure to include different deformations occurring during acquisition.
Sorzano COS, De Isidro-Gomez F, Fernandez-Gimenez E, Herreros D, Marco S, Carazo JM, Messaoudi C
June 23, 2020

Structure-Based Identification and Functional Characterization of a Lipocalin in the Malaria Parasite Plasmodium falciparum

doi: 10.1016/j.celrep.2020.107817
Lipocalin proteins bind small hydrophobic ligands and are involved in various physiological processes like ipid transport and oxidative stress responses. Using crystallography and small-angle X-ray scattering, we show that the lipocalin protein from the malaria parasite Plasmodium falciparum forms a tetrameric structure. It is expressed in the intraerythrocytic stages of the parasite and localizes to the parasitophorous and food vacuoles. Conditional knockdown of impairs parasite development, which can be rescued by treatment with the radical scavenger Trolox or by temporal inhibition of hemoglobin digestion.
Burda PC, Crosskey T, Lauk K, Zurborg A, Söhnchen C, Liffner B, Wilcke L, Pietsch E, Strauss J, Jeffries CM, Svergun DI, Wilson DW, Wilmanns M, Gilberger TW
June 19, 2020

Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy

doi: 10.1007/s10858-020-00328-8
Here we investigate the use of the hanging drop method to grow spheroids which are homogenous in size and shape as a model system to study solid tumors using ssNMR. These spheroids are stable under magic-angle-spinning conditions and show a clear change in metabolic profile as compared to single cell preparations. We utilize DNP-supported ssNMR to show that low concentrations of labelled nanobodies targeting EGFR (7D12) can be detected inside the spheroids.
Damman R, Lucini Paioni A, Xenaki KT, Beltrán Hernández I, van Bergen En Henegouwen PMP, Baldus M
June 12, 2020

NMR quality control of fragment libraries for screening

doi: 10.1007/s10858-020-00327-9
Fragment-based screening evolved as a unique approach for drug discovery in industry and academia. It became a more structure-based approach to inhibitor development and for development of pathway-specific clinical probes. Within iNEXT, an EU-funded H2020 program, a collection of 782 "poised fragments" were assembled to facilitate downstream synthesis of ligands with high affinity by fragment ligation. here, we describe the procedure to assess the quality of the fragment library by NMR spectroscopy. It requires buffer solubility screening, comparison with LC/MS quality control and support by state-of-the-art software for HTP data acquisition and on-the-fly analysis.
Sreeramulu S, Richter C, Kuehn T, Azzaoui K, Blommers MJJ, Del Conte R, Fragai M, Trieloff N, Schmieder P, Nazaré M, Specker E, Ivanov V, Oschkinat H, Banci L, Schwalbe H