Conference Agenda

Overview and details of the sessions of this conference. Please select a date or location to show only sessions at that day or location. Please select a single session for detailed view (with abstracts and downloads if available).

 
 
Session Overview
Session
MS-42: Novel techniques and insights into in vitro and in situ crystallisation for X-ray and electron diffraction.
Time:
Wednesday, 18/Aug/2021:
10:20am - 12:45pm

Session Chair: Lars Redecke
Session Chair: Fasseli Coulibaly
Location: Club A

170 1st floor

Invited: Haruki Hasegawa (USA), Alexandra Ros (USA)


Session Abstract

This microsymposium will aim to present challenges and opportunities in structure determination by crystallography posed by nano, micro and giant crystals. These include new insights into the process of crystallisation and methodological advances that facilitate the production, manipulation and analysis of biological macromolecule crystals, particularly for serial diffraction experiments at microfocus synchrotron and free-electron laser sources, but also for new applications of electron diffraction.

For all abstracts of the session as prepared for Acta Crystallographica see PDF in Introduction, or individual abstracts below.


Introduction
Presentations
10:20am - 10:25am

Introduction to session

Lars Redecke, Fasseli Coulibaly



10:25am - 10:55am

Concurrent crystallization of multiple proteins in a single cell without interfering each other’s phase separation events

Haruki Hasegawa

Amgen Inc, South San Francisco, United States of America

Intracellular protein crystallization occurs in many branches of life, yet the underlying cellular processes remain largely unknown. This is partly because of the scarcity of easily accessible, reproducible recombinant protein models that allow in-depth characterization of intracellular liquid-solid phase separation events. Such limitation prompts the need for identifying various classes of model proteins to examine the similarities, differences, or generalizability of such intracellular crystallization events. Furthermore, to exploit the potential values of cell-made protein crystals and the platforms to produce them, intracellular crystallization should first be understood using diverse classes of model proteins. After validating the individual crystallization events of cellular and viral proteins that readily crystallize in the ER, cytosol or nucleus, I demonstrate up to four independent crystallization events can take place concurrently in various combinations in different subcellular compartments of a single cell. For instance, by co-expressing NEU1 and human IgGs that undergo crystallization or liquid-liquid phase separation in the ER, I demonstrate two independent phase separation events can be simultaneously induced in the same continuous space of the ER lumen without mixing or interfering each other’s phase separation behaviors. Likewise, two concurrent crystallization events can take place in the cytosol or in the nucleus without mixing or interfering each other. Intracellular protein crystallization thus can happen in a crowded physiological cellular environment and does not require high protein purity. Furthermore, I report a simple method to increase the yield of intracellular protein crystals, in terms of crystal size and numbers, by treating the cells with a topoisomerase II inhibitor that blocks cell division without preventing cell size growth. This study not only presents accessible model tools for studying intriguing intracellular protein crystallization events, but also paves a way toward establishing methods and controlling the induction, quality, size, and yield of intracellular protein crystals for high-value proteins.



10:55am - 11:25am

Microfluidic Tools Reducing Sample Amount in Serial Crystallography with XFELs

Alexandra Ros

Arizona State University, Tempe, United States of America

The recent advances of X-ray free electron lasers (XFEL) have enabled serial femtosecond crystallography (SFX) and structure determination for complex proteins such as membrane proteins in high resolution.1-3Importantly, time-resolved (TR) studies have emerged allowing to assess their reaction dynamics. Initial demonstrations focused on light induced reactions; however, a large class of biological macromolecules acts by reaction with specific substrates requiring fast mixing approaches for TR-studies. Microfluidic tools in combination with common liquid injectors for protein crystals allow mixing times in the millisecond to second range, which is suitable to study the dynamics of enzymatic reactions with SFX at XFELs. A large drawback for TR-SFX with substrate-initiated reactions remains the large amount of protein and crystals needed to study the time evolution of a reaction. Every time point to be assessed requires a full data set which multiplies the amount of protein crystals needed by the number of time points to be studied. This may result in unsurmountable protein sample limitations requiring hundreds of mg of protein, which are not attainable for many proteins. Microfluidics allows to tackle this issue by reducing the required amount of protein sample. We propose to inject protein crystals with segmented flow approaches, which deliver crystals to the XFEL only when it pulses. We demonstrate how protein crystals in their mother liquor can be encapsulated in droplets surrounded by an immiscible oil and how these droplets can be intersected with an XFEL using common liquid jet injection methods. We demonstrated this approach reducing the amount of sample required to solve the room temperature structure of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS) at the SPB/SFX instrument at the EuXFEL.4 Furthermore, we demonstrated the ability to electrically trigger the crystal laden droplet release in the microfluidic droplet generator, the interfacing of this approach with miniaturized optical droplet detection and an electronic feedback mechanism to tune the droplet release at a desired frequency matching the repetition rate of a particular XFEL instrument. This approach has been recently tested at the Macromolecular Femtosecond Crystallography instrument at the Linac Coherent Light Source, where the feedback mechanism was successfully implemented. Diffraction was recorded for lysozyme and the protein KDO8PS and the ability to tune the droplet release with a desired delay to the XFEL reference signal was also achieved. This is important to optimize the synchronization with the XFEL when implemented in particular chambers and various geometrical realizations of the droplet generator in relation to the XFEL interaction spot. In follow up experiments, we will assess the amount of sample that is required to obtain a full data set for KDO8PS and couple this strategy with microfluidic mixers, which have already been integrated into the 3D-printed droplet generators. With this approach, we predict that the amount of protein required to achieve a full data set can be reduced by nearly 90 %.

References:

(1) Spence, J. C. H.; Weierstall, U.; Chapman, H. N. Reports on Progress in Physics 2012, 75.

(2) Chapman, H. N.; Fromme, P.; Barty, A., et al. Nature 2011, 470, 73-U81.

(3) Martin-Garcia, J. M.; Conrad, C. E.; Coe, J., et al. Archives of Biochemistry and Biophysics 2016, 602, 32-47.

(4) Echelmeier, A.; Villarreal, J. C.; Kim, D., et al. Nat Comm 2020, 4511.



11:25am - 11:45am

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography

Max T. B. Clabbers2,4, Susannah Holmes1, Timothy W. Muusse3, Parimala Vajjhala3, Sara J. Thygesen3, Alpeshkumar K. Malde5, Dominic J. B. Hunter3,6,7, Tristan I. Croll8, Leonie Flueckiger1, Jeffrey D. Nanson3, Md. Habibur Rahaman3, Andrew Aquila9, Mark S. Hunter9, Mengning Liang9, Chun Hong Yoon9, Jingjing Zhao2, Nadia A. Zatsepin1, Brian Abbey1, Emma Sierecki6, Yann Gambin6, Katryn J. Stacey3, Connie Darmanin1, Bostjan Kobe3,7,10, Hongyi Xu2, Thomas Ve5

1Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia; 2Department of Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden; 3School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia; 4Department of Biological Chemistry, University of California Los Angeles, Los Angeles, California, USA; 5Institute for Glycomics, Griffith University, Southport, Queensland, Australia.; 6EMBL Australia Node in Single Molecule Science, University of New South Wales, Kensington, New South Wales, Australia; 7Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia; 8Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.; 9Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California, USA.; 10Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce proinflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional Xray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higherorder assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX1.

1Clabbers, M., Holmes, S. et.al. MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography, Nature Communications, accepted March 2021, DOI: 10.1038/s41467-021-22590-6

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11:45am - 12:05pm

The crystallomics pipeline, a shotgun approach on native proteomes to (re)discover the unsuspected

Sylvain Engilberge1,2, Olivier Lemaire3, Marie-Caroline Mueller3, Filip Leonarski3, Chia-Ying Huang1, Takashi Tomizaki1, Naohiro Matsugaki5, Antoine Royant4, Vincent Olieric1, Meitian Wang1, Tristan Wagner3

1Swiss Light Source, Paul Scherrer Institut, Forschungsstrasse 111, Villigen-PSI, 5232, Switzerland; 2European Synchrotron Radiation Facility, 38043 Grenoble, France; 3Max-Planck-Institut für Marine Mikrobiologie, Celsiusstraße 1, 28359, Bremen, Germany; 4Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization, Tsukuba, 305-0801, Japan.; 5Institut de Biologie Structurale (IBS), Universite ́ Grenoble Alpes, CEA, CNRS, 71 avenue des Martyrs, Grenoble Cedex 9, 38044, France

Recombinant protein overproduction can lead to aggregation and aberrant artefacts due to the intrinsic specificities of proteins and the requirement of physiological factors (O2 or light-sensitivity, partners, chaperones, cofactors and post-translational modifications requirements). The Wagner’s group (MPI Bremen) has developed a native shotgun approach baptized Crystallomics (Figure) to directly explore native protein complexes from anaerobic microorganisms that contain numerous exotic cofactors (e.g. iron-sulfur cluster).1,2 After protein extraction, the soluble proteome is fractionated through successive chromatography types and the selective process of crystallisation is used as an ultimate purification step. Since this approach targets the most abundant proteins from the soluble fraction, a significant amount of protein crystals representative of the microorganism’s metabolic landscape have been obtained. Ab initio phasing was systematically used for the X-ray structure determination of these unidentified proteins. Crystals were sorted based on their colours. Sulfur-SAD3,4 were performed on the transparent crystals by collecting high multiplicity and multi-orientations data at low energies on X06DA at the Swiss Light Source or on BL-1A at KEK. To reduce noise and improve the accuracy of the data quality some of the protein crystals were shaped with a deep-UV laser (to decrease X-ray absorption) and diffraction experiments were performed under helium environment with the recently developed PSI JUNGFRAU detector.5 For coloured crystals containing their native cofactors and heavy elements, X-ray fluorescent spectra were systematically measured. SAD were then performed at the edge of the atom of interest. Protein targets were then identified either by manual sequencing in the electron density maps or by fold similarity after reconstruction of a poly-alanine model.In complement to X-ray diffraction, in cristallo UV/vis absorption spectra were recorded by using the microspectrophotometer icOS6 at the ESRF to further investigate the nature of the state adopted by metal/absorbing centers. This synergistic approach proved that crystallisation not only separates proteins from each other but is also a powerful tool to isolate and characterized different protein states from a mixture.

[1] Vögeli B, Engilberge S, Girard E, Riobé F, Maury O, Erb TJ, Shima S, Wagner T. (2018) Proc. Natl. Acad. Sci. U S A. 27, 3380-3385.

[2] Engilberge S., Wagner T., Santoni G., Breyton C., Shima S., Franzetti B., Riobé F., Maury O., Girard E. (2019) J. Appl. Cryst.28, 722-731.

[3] Olieric V., Weinert T., Finke A. D., Anders C., Li D., Olieric N., Borca C.N., Steinmetz M.O., Caffrey M., Jinek M., Wang M. (2016) Acta Cryst. D72, 421-429.

[4] Basu S, Olieric V, Leonarski F, Matsugaki N, Kawano Y, Takashi T, Huang CY, Yamada Y, Vera L, Olieric N, Basquin J, Wojdyla JA, Bunk O, Diederichs K, Yamamoto M, Wang M. (2019) IUCrJ. 6, 373-386.

[5] Leonarski F, Redford S, Mozzanica A, Lopez-Cuenca C, Panepucci E, Nass K, Ozerov D, Vera L, Olieric V, Buntschu D, Schneider R, Tinti G, Froejdh E, Diederichs K, Bunk O, Schmitt B, Wang M. (2018) Nat. Methods.15, 799-804.

[6] von Stetten D, Giraud T, Carpentier P, Sever F, Terrien M, Dobias F, Juers DH, Flot D, Mueller-Dieckmann C, Leonard GA, de Sanctis D and Royant A. In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF. (2015) Acta Crystallogr. D, 71, 15-26.



12:05pm - 12:25pm

Protein crystallization assisted by the crystallophore.

Eric Girard1, Zaynab Alsalman1, Adeline Robin1, Sylvain Engilberge1, Amandine Roux2, François Riobé2, Olivier Maury2

1Institut de Biologie Structurale, Grenoble, France; 2Ecole Normale Supérieure, Lyon, France

Obtaining crystals and solving the phase problem remain major hurdles encountered by bio-crystallographers in their race to get new high-quality structures. The crystallophore, Xo4, is a family of nucleating and phasing molecules based on lanthanide complexes. Tb-Xo4 was the first molecule of this family to be described [1].

Results obtained on more than fifteen proteins will be described and will show that Tb-Xo4 is an efficient tool to promote protein crystallization. Among these results, we will show that (i) Tb-Xo4 increases the number of crystallization conditions by promoting unique ones [1,2] (ii) the crystalline forms promoted by the crystallophore bypass crystal defects often encountered by crystallographers such as low-resolution diffracting samples or crystals with twinning [3] and (iii) the crystallization reproducibility is largely improved, a particular issue in structure-based drug design.

Contrary to the dogma that crystallization can only be promoted from pure protein sample, we have shown that crystals can be obtained from enriched fractions containing several proteins [3] leading to the structure determination of a protein complex [4]. Even more unexpected, the crystallophore is able to induce crystallization directly from the protein solution, as exemplified by the crystallization of hen egg white lysozyme in water [5].

Finally, we will also present preliminary results on several crystallophore variants showing complementarity with Tb-Xo4 thus enlarging the success in defining exploitable crystallization conditions.

Altogether, crystallophore is an efficient solution for protein crystallization and structure determination in the bio-crystallographer toolbox.

[1] Engilberge, S., Riobé, F., Di Pietro, S., Lassalle, L., Coquelle, N., Arnaud, C.-A., Pitrat, D., Mulatier, J.-C., Madern, D., Breyton, C., Maury, O. & Girard, E. (2017). Chem. Sci. 8, 5909–5917.[2] Jiang, T., Roux, A., Engilberge, S., Alsalman, Z., Di Pietro, S., Franzetti, B., Riobé, F., Maury, O. & Girard, E. (2020). Crystal Growth & Design. 20, 5322–5329.[3] Engilberge, S., Wagner, T., Santoni, G., Breyton, C., Shima, S., Franzetti, B., Riobé, F., Maury, O. & Girard, E. (2019). Journal of Applied Crystallography. 52, 722–731.[4] Vögeli, B., Engilberge, S., Girard, E., Riobé, F., Maury, O., Erb, T. J., Shima, S. & Wagner, T. (2018). Proceedings of the National Academy of Sciences. 115, 3380–3385.[5] de Wijn, R., Rollet, K., Engilberge, S., McEwen, A. G., Hennig, O., Betat, H., Mörl, M., Riobé, F., Maury, O., Girard, E., Bénas, P., Lorber, B. & Sauter, C. (2020). Crystals. 10, 65.

Keywords: Crystallisation; crystallophore; nucleating agents; structure determination; phasing.

Authors acknowledge financial supports from the Fondation Maison de la Chimie, Agence Nationale de la Recherche (ANR Ln23-13-BS07-0007-01) and Region AuRA for (program Xo4-2.0).



12:25pm - 12:45pm

Protein crystallisation in agarose gel, a cheap and versatile technique

Jose A. Gavira1, Fiora Artusio2, Albert Castellví3, Roberto Pisano2

1CSIC, Granada, Spain; 2Politecnico di Torino, Torino, Italy; 3Structural Biology, Molecular Biology Institute of Barcelona, Barcelona, Spain

Crystallization in hydrogels is not a frequent practice in bio-crystallography, although the benefits are multiple: prevents convection and crystal sedimentation, acts as impurity filter, etc., and have been proven to be the cheapest means to produce protein crystals of high quality similar to those obtained under microgravity conditions [1-2]. Moreover, gel grown protein crystals are excellent candidates as seeds to produce crystals of bigger size for neutron diffraction or as media for crystals delivery in serial femtosecond crystallography [3].

Hydrogel should also be considered to exert control over the nucleation and growth processes. In this work we will present our most recent studies on the influence of agarose over the nucleation and growth of protein crystals. Crystal number and size was successfully tuned in a wide range of agarose concentration while keeping constant other conditions. Using five model proteins we demonstrate that the influence of gel content is independent of the protein nature, allowing the mathematical prediction of crystals flux and size with little experimental effort. The convection free environment obtained even at low agarose concentration [4] permits the obtention of high homogeneous micro-crystals slurries (Figure 1) that could be used for serial crystallography application [3] or for the mass production of enzyme crystals for industrial application [5]. Last, we will also show how it allows to explore the phase diagram under a kinetic regime that may facilitate the growth of different polymorphs.

Figure 1. Crystal size and number are fine-tuned using agarose as non-convective media during the crystallization process. (a) Proteinase-K crystals size as a function of agarose concentration and (b) as a function of precipitant concentration at fix agarose concentration of 0.1% (w/v). Scale bar is 100 µm.

[1] Gavira, J. A., Otálora, F., González-Ramírez, L. A., Melero, E., Driessche, A. E. S. v., & García-Ruíz, J. M. (2020). Crystals, 10, 68.[2] Lorber, B.; Sauter, C.; Théobald-Dietrich, A.; Moreno, A.; Schellenberger, P.; Robert, M.C.; Capelle, B.; Sanglier, S.; Potier, N.; Giegé, R. (2009). Prog. Biophys. Mol. Biol. 101, 13.[3] Artusio, F.; Castellví, A.; Sacristán, A.; Pisano, R.; Gavira, J.A. (2020). Cryst. Growth Des., 20, 5564.[4] Garcia-Ruiz, J.M.; Novella, M.; Moreno, R.; Gavira, J.A. (2001). J. Cryst. Growth, 232, 165.[5] Fernández-Penas, R.; Verdugo-Escamilla, C.; Martínez-Rodríguez, S.; Gavira, J.A. (2021). Cryst. Growth Des., 21, 1698.

Keywords: agarose hydrogel; protein crystal nucleation, serial crystallography

Supported by project BIO2016-74875-P (MINECO), Spain co-funded by the Fondo Europeo de Desarrollo Regional (FEDER funds), European Union.