The Stahlberg group has built a suite of programs to process cryo-electron microscopy images. The software system 2dx [1] was able to fully automatically process cryo-EM images of two-dimensional membrane protein crystals, resulting in a high-resolution 3D structure of the membrane protein while data are being collected. 2dx is based on the MRC software suite and was extended by additional programs, e.g., to apply single particle and maximum likelihood algorithms to 2D crystal geometries. We have further developed FOCUS [2], which establishes an interface between cryo-EM data collection and data processing. FOCUS monitors a cryo-EM instrument, and once an image or a movie or tilt series is recorded, FOCUS will submit it to a fully automated and user-adaptable processing pipeline. Daniel Castano in our group has also developed the DYNAMO software package [3] for sub-volume analysis of Cryo-electron tomography datasets. DYNAMO is now widely used by a large user community [1] Gipson, B., Zeng, X., Zhang, Z. Y., & Stahlberg, H. (2007). 2dx-User-friendly image processing for 2D crystals. Journal of Structural Biology, 157(1), 64&72.
[2] Biyani, N., Righetto, R. D., McLeod, R., Caujolle-Bert, D., Castano-Diez, D., Goldie, K. N., & Stahlberg, H. (2017). Focus: The interface between data collection and data processing in cryo-EM. Journal of Structural Biology, 198(2), 124&133.
[3] Castaño-Díez, D., Kudryashev, M., Arheit, M., & Stahlberg, H. (2012). Dynamo: A flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. Journal of Structural Biology, 178(2), 139& 151.

We have studied the brain tissue obtained after autopsy of patients who died from Parkinson's disease or other synucleinopathies. We identified the large alpha-synuclein-rich aggregates in brain tissue (Lewy bodies), but surprisingly found by correlative light and electron microscopy (CLEM) that these in the majority of cases are primarily composed of densely packed membrane fragments, with aSyn fibrils contributing only in same cases a minor fraction [1]. In contrast, the brain of Multiple System Atrophy (MSA) patients contains aSyn-rich GCI aggregates that indeed are composed of fibrillar material. We also developed and implemented tools to study the structure of human neurodegenerative brain tissue and applied these to various neurodegenerative tissues [2-4]. [1] Lewy pathology in Parkinson's disease consists of a crowded organellar membranous medley
Shahmoradian, S., Lewis, A., Genoud, C., Graff, A., Pérez Navarro, P., Goldie, K., Sütterlin, R., Castano-Diez, D., Ingrassia, A., Rozemuller, A.J.M., Paepe, A.D., Erny, J.Staempfli, A., Hoernschemeyer, J., Niedieker, D., El-Mashtoly, S.F., Gerwert, K., Bohrmann, B., Britschgi, M., Stahlberg, H.*, van de Berg, W.D.J.*, Lauer, M.E.*
Nature Neuroscience
22, 1099-1109 (2019)
*=corresponding authors
[2] Alterations in sub-axonal architecture between normal aging and Parkinson's diseased human brains using label-free cryogenic X-ray nanotomography
Tran, H.T., Tsai, E.H.R., Lewis, A., Moors, T., Bol, J.G.J.M., Rostami, I., Diaz, A., Joker, A.J., Guizar-Sicairos, M., Raabe, J., Stahlberg, H., van de Berg, W.D.J., Holler, M., Shahmoradian, S.
Frontiers in Neuroscience 14 (570019), (2020)
[3] Imaging of post-mortem human brain tissue using electron and X-ray microscopy
Lewis, A., Genoud, C., Pont, M., van de Berg, W.D.J., Frank, S., Stahlberg, H., Shahmoradian, S., Al-Amoudi, A.
Current Opinion Structural Biology 58, 138-148 (2019)
[4] Cerebral Corpora amylacea are dense membranous labyrinths containing structurally preserved cell organelles
Pérez Navarro, P., Genoud, C., Castano-Diez, D., Graff-Meyer, A., Lewis, A., de Gier, Y., Lauer, M.E., Britschgi, M., Bohrmann, B., Frank, S., Hench, J., Schweighauser, G., Rozemuller, A.J.M., van de Berg, W.D.J., Stahlberg, H.*, Shahmoradian, S.*
Scientific Reports 8(18046), (2018).
*=corresponding authors

Our group has also studied fibrils of aSyn, which were grown in-vitro de-novo or after seeding with human brain sample (PMCA). We have determined the atomic structures of several aSyn fibril conformations (prionoid strains) [1-4]. [1] New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy
Guerrero-Ferreira, R., Kovacik, L., Ni, D., Stahlberg, H.
Current Opinion in Neurobiology 61, 89-95 (2020)
[2] Two new polymorphic structures of alpha-synuclein solved by cryo-electron microscopy
Guerrero-Ferreira, R., Taylor, N., Arteni, A.-A., Kumari, P., Mona, D., Ringler, P., Britschgi, M., Lauer, M.E., Verasdock, J., Riek, R., Melki, R., Meier, B.H., Böckmann, A., Bousset, L., Stahlberg, H.
eLife 8:e48907, (2019)
[3] Cryo-EM structure of alpha-synuclein fibrils
Guerrero-Ferreira, R., Taylor, N., Mona, D., Ringler, P., Lauer, M. E., Riek, R., Britschgi, M., Stahlberg, H.
eLife 7(e36402), (2018)
[4] Cryo-EM structure of alpha-synuclein fibrils amplified by PMCA from PD and MSA patient brains
Burger, D., Fenyi, A., Bousset, L., Stahlberg, H., Melki, R.
bioRxiv 2021.07.08.451588, (2022)

Our lab has developed microfluidics-based sample preparation for cryo-EM. The cytosol of a single, hand-picked neuronal cell or small nanoliter volumes of sample can be aspired with a capillary. Target proteins can optionally be purified via magnetic beads coupled to FAB antibody fragments from the sample. Finally, the miniature quantities of sample can be written in a loss-less manner onto a cryo-EM grid. The toolchain developed by Dr. Thomas Braun and student co-workers in the Stahlberg lab allowed to aspire < 1 microliter of HeLa cell cytosol, purify endogenous 20S proteasome sample out of that, and write the resulting 9 nanoliters of pure protein onto a cryo-EM grid, which allowed us to determine the atomic structure of the human 20S proteasome [1]. The patents from this work are now licensed to CryoWrite AG (Basel, Switzerland), who offers this technology as a commercial instrument: The CryoWriter. [1] Schmidli, C., Albiez, S., Rima, L., Righetto, R., Mohammed, I., Oliva, P., Kovacik, L., Stahlberg, H., & Braun, T. (2019). Microfluidic protein isolation and sample preparation for high-resolution cryo-EM. Proceedings of the National Academy of Sciences of the United States of America, 116(30), 15007&15012.

Our lab has implemented 4D-STEM with Ptychography data processing at high throughput on a prototype TFS Titan Krios electron microscope, equipped with a probe aberration corrector and a high-speed (120'000 frames per second) Dectris Arina camera. 4D-STEM Ptychography reconstructions of single particles and tissue sections can now be obtained at much improved contrast compared to conventional cryo-EM [1]. [1] Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution
Küçükoglu, B., Mohammed, I., Guerrero Ferreira, R., Ribet, S.M., Varnavides, G., Leidl, M.L., Lau, K., Nazarov, S., Myasnikov, A., Kube, M., Radecke, J., Sachse, C., Müller-Caspary, K., Ophus, C., Stahlberg, H.
Nature Communications, (2024)