PC

Available Instruments

• VP ITC, MicroCal - Isothermal Titration Calorimetry, study of biomolecular interactions in solution
• PEAQ-ITC, MicroCal - Isothermal Titration Calorimetry, study of biomolecular interactions in solution
• Monolith NT.115 (Microscale thermophoresis), NanoTemper - measuring binding affinities (labelling of 1 partner required)
• Monolith NT. Label-free (Microscale thermophoresis), NanoTemper - measuring binding affinities (label-free)
• DLS Nanostar (Dynamic light scattering), Malvern - particle size measurements. Addressing dispersity/ aggregational state of samples.
• Biacore T200, GE - SPR, interaction of biomolecules and kinetics
• CD (Circular Dichroism), Applied Photophysics - Secondary structure analysis.
• Prometheus NT.48 with backscattering optics (nano-Differential Scanning Fluorimetry technology), NanoTemper - study protein stability and protein aggregation.
• FTIR (Fourier Transform Infrared Spectrometer) Bruker - Protein spectrum. Analysing protein signals from formulation components.
• MALDI TOF, Bruker/CovalX - Determination of the molecular mass of protein samples and peptides resulting from proteolytic digestions. CovalX HM4 High-Mass System enables protein complex analysis and intact protein analysis.
• Octet RED96 System, PAL ForteBio - 96-well characterization of protein-protein and protein-small molecule binding kinetics, determination of protein concentrations.
• SPARK 20M Plate reader, TECAN - Detection Absorbance, Fluorescence intensity, FP, TRF, FRET, TR-FRET, luminescence (flash, glow, multi-colour), Alpha Technology, Automated live cell imaging
• Stopped-Flow, Applied Photophysics - Time resolved experiments
• Mass photometer, Refeyn - can monitor protein-protein interactions at a single-molecule level with high-sensitivity and can at the same time determine molecular weight of proteins and protein complexes with a high dynamic range and great accuracy. The mass photometer is an ideal tool for quality control in the protein structural analysis workflow as it can assess the molecular mass and the oligomerisation status of a sample in one measurement.

• SSM-based electrophysiology, SURFE²R N1 - designed for the measurements of electrogenic transporters (symporters, exchangers and uniporters) and pumps. Usually these proteins have low turnover rates compared to ion channels. SURFE²R technology compensates for that with a large sensor size which allows for the measurement of up to 109 transporters at the same time to yield the best signal to noise ratio.

Services

We support users with the design, execution and data analysis of biophysical experiments aimed at the characterization of proteins, protein complexes and interactions between proteins and other types of molecules.

Further Information

Our protein characterization platform located at the CSSB includes several biophysical instruments available for in-house and external users. We offer services for protein quality control in addition to characterization techniques for studying the interaction of biomolecules. Techniques available: MS MALDI coupled to a high mass detector, Thermofluor (TF), Circular Dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), Isothermal Titration Calorimetry (ITC), nano Differential Scanning Fluorimetry (nDSF), Surface Plasmon Resonance (SPR), Biolayer interferometry (Octet), Microscale Thermophoresis (MST) and Dynamic Light Scattering (DLS).

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HTX

Available Instruments

• Mosquito LCP (two units)
• Scorpion Screen Formulator
• Biomec 4000 (liquid handling platform)
• Rock Imager at 4 and 19 °C
• Automatic crystal harvester (CrystalDirect)

Services

 The HTX facility offers a variety of services related to protein crystallisation:

• Automatic setup of crystallization trays using Mosquito-LCP
• Low volume crystallisation screening for sitting and hanging drop experiments
• >30 commercial screens available
• Crystal condition optimisation
• Large storage capacity at 19ºC and 4ºC
• Specialised Crystal information management system (CRIMS) to schedule and monitor experiments.
• Automated image acquisition (Vis/UV) and remote access environment for the user via the CRIMS system.

Further Information

Our high-throughput crystallization laboratory offers initial crystallization screens from various suppliers as well as customized screens for optimization of initial hits, both suitable for soluble and membrane proteins. Drops are set with nanolitre dispensing robots; the TTP’s MOSQUITO equipped with an LCP (Lipidic Cubic Phase) module. Online observation of the plates is possible via the Crystallization Information Management System (CRIMS), which makes results available to users in real-time, along with all experimental parameters. We offer the CrystalDirect® technology, able to harvest protein crystals from 96-well CrystalDirectTM plates by means of laser photoablation. We facilitate access to Biological SAXS measurements with near-real time outputs of structural parameters and low-resolution solution-state structures in collaboration with the SAXS group.

Access & Team

Access

The PC facility is a fee-for-service (non-profit) technology platform operated by EMBL in Hamburg. No service fees are charged for usage of this equipment by EMBL, CSSB and beamline users. An online system (iLAB) to streamline the process of booking instruments, ordering and billing core service requests is in place and in routine use.

All users must follow a safety training before working in our laboratories. Complete the SPC safety briefing and examination before your visit though SMIS. Please go to the Safety tab and select "Standard Training"

Our acknowledgement policy

Please acknowledge any substantial contributions of the SPC Facility to your research in publications. Please state the following in the acknowledgement section of your publication: “We acknowledge technical support by the SPC facility at EMBL Hamburg”.

Please don't forget to let us know when you publish papers that acknowledge us. This will help us keep track and to justify the existence of the facility to funding bodies.

Transnational Access Programme

All European researchers (academic and industry) can apply for transnational access to perform their Structural Biology research at the SPC facility.

Team

12

PhD Student

Osvaldo Burastero
Phone:+49 40 8998 87568

PostDoc

Lucas Defelipe
Phone:+49 40 8998 87566

E-Mail

PhD Student

George Draper-Barr
Phone:+49 40 8998 87566

Facility Head

Dr. Maria Garcia Alai
Phone:+49 040 8990 2145

E-Mail

PhD Student

Tatiana Kupriianova
Phone:+49 40 8998 87566

Staff Scientist

Dr. Stephan Niebling
Phone:+49 40 8998 87655

E-Mail

TA

David Ruiz-Carillo

Trainee

Janina Schiller

Staff Scientist

Angelica Struve Garcia
Phone:+49 40 8998 87655

E-Mail

Trainee

Thorben Thalacker
Phone:+49 40 8998 87566

PostDoc

Maria Agustina Toscanini

TA

Katharina Veith
Phone:+49 40 8998 87599

Address

CSSB Centre for Structural Systems Biology
c/o Deutsches Elektronen-Synchrotron DESY
Notkestraße 85, Building 15
D-22607 Hamburg

Facility Operator: European Molecular Biology Laboratory (EMBL)

Publications

2024

Lis K, Plewka J, Menezes F, Bielecka E, Chykunova Y, Pustelny, K, Niebling S, Garcia AS, Garcia-Alai M, Popowicz GM, Czarna A, Kantyka T, Pyrc K (2024) Sars-cov-2 mpro oligomerization as a potential target for therapy. Int J Biol Macromol. 276(Pt 1):133473. doi: 10.1016/j.ijbiomac.2024.133473

Defelipe, L., Veith, K., Burastero, O., Kupriianova, T., Bento, I., Skruzny, M., Kölbel, K., Uetrecht, C., Thuenauer, R., & Garcia-Alai, M. M. (2024). Subtleties in Clathrin Heavy Chain Binding Boxes provide selectivity among Adaptor Proteins of Budding Yeast. Nat Commun 15, 9655. https://doi.org/10.1038/s41467-024-54037-z

Draper-Barr, G. Defelipe L., Ruiz Carrillo D., Gustavsson E., Landau M. and Garcia-Alai M. (2024). Sla2 is a core interaction hub for Clathrin Light Chain and the Pan1/End3/Sla1 Complex. bioRxiv. doi: https://doi.org/10.1101/2024.11.14.623549

2023

Niebling S, Burastero O, García-Alai M (2023) Biophysical characterization of membrane proteins. Advanced Methods in Structural Biology. Methods in Molecular Biology, vol 2652. https://doi.org/10.1007/978-1-0716-3147-8_12

2022

Hassani Nia F, Woike D, Bento I, Niebling S, Tibbe D, Schulz K, Hirnet D, Skiba M, Hönck HH, Veith K, Günther C, Scholz T, Bierhals T, Driemeyer J, Bend R, Failla AV, Lohr C, Alai MG, Kreienkamp HJ (2022) Structural deficits in key domains of Shank2 lead to alterations in postsynaptic nanoclusters and to a neurodevelopmental disorder in humans. Mol Psychiatry https://doi.org/10.1038/s41380-022-01882-3

Sander S, Müller I, Alai MG, Nicke A, Tidow H (2022) New insights into P2X7 receptor regulation: Ca2+-calmodulin and GDP bind to the soluble P2X7 ballast domain. J Biol Chem. 102495. doi: 10.1016/j.jbc.2022.102495

Srinivasan V, Brognaro H, Prabhu PR, de Souza EE, Günther S, Reinke PYA, Lane TJ, Ginn H, Han H, Ewert W, Sprenger J, Koua FHM, Falke S, Werner N, Andaleeb H, Ullah N, Franca BA, Wang M, Barra ALC, Perbandt M, Schwinzer M, Schmidt C, Brings L, Lorenzen K, Schubert R, Machado RRG, Candido ED, Oliveira DBL, Durigon EL, Niebling S, Garcia AS, Yefanov O, Lieske J, Gelisio L, Domaracky M, Middendorf P, Groessler M, Trost F, Galchenkova M, Mashhour AR, Saouane S, Hakanpää J, Wolf M, Alai MG, Turk D, Pearson AR, Chapman HN, Hinrichs W, Wrenger C, Meents A, Betzel C (2022) Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease. Commun Biol. 5(1):805. doi: 10.1038/s42003-022-03737-7

Ewert W, Günther S, Miglioli F, Falke S, Reinke PYA, Niebling S, Günther C, Han H, Srinivasan V, Brognaro H, Lieske J, Lorenzen K, Garcia-Alai MM, Betzel C, Carcelli M, Hinrichs W, Rogolino D, Meents A (2022) Hydrazones and Thiosemicarbazones Targeting Protein-Protein-Interactions of SARS-CoV-2 Papain-like Protease. Front Chem. 10:832431. doi: 10.3389/fchem.2022.832431

Dirscherl C, Löchte S, Hein Z, Kopicki JD, Harders AR, Linden N, Karner A, Preiner J, Weghuber J, Garcia-Alai M, Uetrecht C, Zacharias M, Piehler J, Lanzerstorfer P, Springer S (2022) Dissociation of β2m from MHC class I triggers formation of noncovalent transient heavy chain dimers.  J Cell Sci. 135(9):jcs259489. doi: 10.1242/jcs.259498.

Bücker R, Seuring C, Cazey C, Veith K, García-Alai M, Grünewald K, Landau M (2022) The Cryo-EM structures of two amphibian antimicrobial cross-β amyloid fibrils. Nat Commun. 13(1):4356. doi: 10.1038/s41467-022-32039-z

Burastero O, Defelipe LA, Gola G, Tateosian NL, Lopez ED, Martinena CB, Arcon JP, Traian MD, Wetzler DE, Bento I, Barril X, Ramirez J, Marti MA, Garcia-Alai MM, Turjanski AG (2022) Cosolvent Sites-Based Discovery of Mycobacterium Tuberculosis Protein Kinase G Inhibitors. Journal of medicinal chemistry. 65(14):9691-9705. doi:10.1021/acs.jmedchem.1c02012

Niebling S, Veith K, Vollmer B, Lizarrondo J, Burastero O, Schiller J, Struve García A, Lewe P, Seuring C, Witt S, García-Alai M (2022) Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins. Front. Mol. Biosci. 9:882288. doi: 10.3389/fmolb.2022.882288

Kopicki JD, Saikia A, Niebling S, Günther C, Anjanappa R, Garcia-Alai M, Springer S, Uetrecht C (2022) Opening opportunities for Kd determination and screening of MHC peptide complexes. Commun Biol. 5(1):488. doi: 10.1038/s42003-022-03366-0.

2021

Lorenzo R, Defelipe LA, Aliperti L, Niebling S, Custódio TF, Löw C, Schwarz JJ, Remans K, Craig PO, Otero LH, Klinke S, García-Alai M, Sánchez IE, Alonso LG (2021) Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif. J Biol Chem.101175. doi: 10.1016/j.jbc.2021.101175.

López-Méndez B, Baron B, Brautigam CA, Jowitt TA, Knauer SH, Uebel S, Williams MA, Sedivy A, Abian O, Abreu C, Adamczyk M, Bal W, Berger S, Buell AK, Carolis C, Daviter T, Fish A, Garcia-Alai M, Guenther C, Hamacek J, Holková J, Houser J, Johnson C, Kelly S, Leech A, Mas C, Matulis D, McLaughlin SH, Montserret R, Nasreddine R, Nehmé R, Nguyen Q, Ortega-Alarcón D, Perez K, Pirc K, Piszczek G, Podobnik M, Rodrigo N, Rokov-Plavec J, Schaefer S, Sharpe T, Southall J, Staunton D, Tavares P, Vanek O, Weyand M, Wu D (2021) Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study. Eur Biophys J. 50(3-4):411-427. doi:10.1007/s00249-021-01532-6.

de Marco A, Berrow N, Lebendiker M, Garcia-Alai M, Knauer SH, Lopez-Mendez B, Matagne A, Parret A, Remans K, Uebel S, Raynal B (2021) Quality control of protein reagents for the improvement of research data reproducibility. Nat Commun.12(1):2795. doi:10.1038/s41467-021-23167-z.

Niebling S, Burastero O, Bürgi J, Günther C, Defelipe LA, Sander S, Gattkowski E, Anjanappa R, Wilmanns M, Springer S, Tidow H, García-Alai M (2021) FoldAffinity: binding affinities from nDSF experiments. Sci Rep. 11(1):9572. doi:10.1038/s41598-021-88985-z.

Berrow N, de Marco A, Lebendiker M, Garcia-Alai M, Knauer SH, Lopez-Mendez B, Matagne A, Parret A, Remans K, Uebel S, Raynal B (2021) Quality control of purified proteins to improve data quality and reproducibility: results from a large-scale survey. Eur Biophys J. 50(3-4):453-460. doi:10.1007/s00249-021-01528-2.

Winkel Missel J, Salustros N, Ramos Becares E, Hyld Steffen J, Gerdt Laursen A, Struve Garcia A, Garcia-Alai MM, Kolar Č, Gourdon P, Gotfryd K (2021) Cyclohexyl-α maltoside as a highly efficient tool for membrane protein studies. Current Research in Structural Biology. https://doi.org/10.1016/j.crstbi.2021.03.002.

Lizarrondo J, Klebl DP, Niebling S, Abella M, Schroer MA, Mertens HDT, Veith K, Thuenauer R, Svergun DI, Skruzny M, Sobott F, Muench SP, Garcia-Alai MM (2021) Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis. Nat Commun.12(1):2889. doi: 10.1038/s41467-021-23151-7.

Günther S, Reinke PYA, Fernández-García Y, Lieske J, Lane TJ, Ginn HM, Koua FHM, Ehrt C, Ewert W, Oberthuer D, Yefanov O, Meier S, Lorenzen K, Krichel B, Kopicki JD, Gelisio L, Brehm W, Dunkel I, Seychell B, Gieseler H, Norton-Baker B, Escudero-Pérez B, Domaracky M, Saouane S, Tolstikova A, White TA, Hänle A, Groessler M, Fleckenstein H, Trost F, Galchenkova M, Gevorkov Y, Li C, Awel S, Peck A, Barthelmess M, Schluenzen F, Lourdu Xavier P, Werner N, Andaleeb H, Ullah N, Falke S, Srinivasan V, França BA, Schwinzer M, Brognaro H, Rogers C, Melo D, Zaitseva-Doyle JJ, Knoska J, Peña-Murillo GE, Mashhour AR, Hennicke V, Fischer P, Hakanpää J, Meyer J, Gribbon P, Ellinger B, Kuzikov M, Wolf M, Beccari AR, Bourenkov G, von Stetten D, Pompidor G, Bento I, Panneerselvam S, Karpics I, Schneider TR, Garcia-Alai MM, Niebling S, Günther C, Schmidt C, Schubert R, Han H, Boger J, Monteiro DCF, Zhang L, Sun X, Pletzer-Zelgert J, Wollenhaupt J, Feiler CG, Weiss MS, Schulz EC, Mehrabi P, Karničar K, Usenik A, Loboda J, Tidow H, Chari A, Hilgenfeld R, Uetrecht C, Cox R, Zaliani A, Beck T, Rarey M, Günther S, Turk D, Hinrichs W, Chapman HN, Pearson AR, Betzel C, Meents A. (2021) X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease. Science. 2021 Apr 2:eabf7945. doi: 10.1126/science.abf7945.

2020

Heidemann J, Kölbel K, Konijnenberg A, Van Dyck J, Garcia-Alai M, Meijers R, Sobott F, Uetrecht C. (2020) Further insights from structural mass spectrometry into endocytosis adaptor protein assemblies. Int J Mass Spectrom. 2019 Oct 18;447:116240. doi: 10.1016/j.ijms.2019.116240.

Anjanappa R, Garcia-Alai M, Kopicki JD, Lockhauserbäumer J, Aboelmagd M, Hinrichs J, Nemtanu IM, Uetrecht C, Zacharias M, Springer S, Meijers R (2020) Structures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection. Nat Commun. 11;11(1):1314. doi: 10.1038/s41467-020-14862-4.

2019

Saini SK, Tamhane T, Anjanappa R, Saikia A, Ramskov S, Donia M, Svane IM, Jakobsen SN, Garcia-Alai M, Zacharias M, Meijers R, Springer S, Hadrup SR (2019) Empty peptide-receptive MHC class I molecules for efficient detection of antigen-specific T cells. Sci Immunol 410.1126/sciimmunol.aau9039 doi: 10.1126/sciimmunol.aau9039

Moritz A, Anjanappa R, Wagner C, Bunk S, Hofmann M, Pszolla G, Saikia A, Garcia-Alai M, Meijers R, Rammensee HG, Springer S, Maurer D (2019) High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules. Sci Immunol 410.1126/sciimmunol.aav0860. doi: 10.1126/sciimmunol.aav0860.

Kotov V, Bartels K, Veith K, Josts I, Subhramanyam UKT, Gunther C, Labahn J, Marlovits TC, Moraes I, Tidow H, Low C, Garcia-Alai MM (2019) High-throughput stability screening for detergent-solubilized membrane proteins. Sci Rep 9: 10379.doi: 10.1038/s41598-019-46686-8

Gattkowski E, Johnsen A, Bauche A, Möckl F, Kulow F, Garcia Alai M, Rutherford TJ, Fliegert R, Tidow H. (2019) Novel CaM-binding motif in its NudT9H domain contributes to temperature sensitivity of TRPM2. Biochim Biophys Acta Mol Cell Res. 2019 Jul;1866(7):1162-1170. doi: 10.1016/j.bbamcr.2018.12.010.

2018

Garcia-Alai MM, Heidemann J, Skruzny M, Gieras A, Mertens HDT, Svergun DI, Kaksonen M, Uetrecht C, Meijers R (2018) Epsin and Sla2 form assemblies through phospholipid interfaces. Nat Commun 9: 328 doi: 10.1038/s41467-017-02443-x

Molina IG, Josts I, Almeida Hernandez Y, Esperante S, Salgueiro M, Garcia Alai MM, de Prat-Gay G, Tidow H. (2018) Structure and stability of the Human respiratory syncytial virus M 2-1 RNA-binding core domain reveals a compact and cooperative folding unit. Acta Crystallogr F Struct Biol Commun. 2018 Jan 1;74(Pt 1):23-30. doi: 10.1107/S2053230X17017381.

Tools

eSPC, an Online Data Analysis Platform for Molecular Biophysics

The eSPC platform provides easy to use software for the understanding of biophysical experiments.

https://spc.embl-hamburg.de/