Protein Structure determination using cryo-electron microscopy

My group is focusing on the structural studies of polyketide synthase, non-ribosomal peptide synthase, pore forming toxin, bacterial secretion system, bacterial ion channels and GPCRs using cryo-electron microscopy. Single particle cryo-electron microscopy (cryo-EM) techniques are developing rapidly for the structural characterization of complicated biological complexes. These methods generate snapshots of macromolecules in solution and under highly controlled biochemical environments, a crucial condition for probing physiological structures of distinct functional states. Recent high impact publications suggest that cryo-EM has enough potential to resolve the high resolution structure of biological samples and is an essential structural biology instrument, which allows us to determine the three-dimensional (3D) structures of large biological macromolecules, macromolecular complexes, and cell components We use different biochemical, biophysical techniques including negative staining electron microscopy and cryo-EM techniques to characterize the conformational dynamics of very small (100 kDa) protein complexes to very large biological macromolecules (2.6 MDa).

Lab news

• Nayanika won second prize for oral presentation in MBU symposium, 2019

• Anil won first prize for poster presentation in EMSI, 2019

• Haaris Ahsan Safdari and Ishika Pramanick, PhD student of our lab won the second and consolation prize respectively in the International Conference on Microscope and XXXIX Annual Meeting of Electron Microscope Society of India, Bhubaneshwar, India (2018)

• Early Career research Award of DST, 2017

• DBT Advanced Centre for Cryo-electron microscopy (Cryo-EM), 2017

• Somnath Dutta received Ramanujan Fellowship, 2016

Research interest

Pore-forming toxins (PFTs) are the most common bacterial cytotoxic proteins that

cause lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. PFTs are mostly water-soluble toxin and the toxin monomer self-assembles on the target cell surface to the more stable oligomer, which inserts into the membrane bilayer to form a diffusion channel. Extensive

research effort has been devoted to understanding the molecular pore formation mechanisms and the functions of certain model PFTs. Cryo-EM has been recently revolutionized by the employment of direct

electron detector (DED). This can facilitate the calculation of 3D structure of PFTs at close to atomic resolution, which might shed light on the molecular mechanisms and the functions of PFTs.

Bacteria use a broad variety of membrane transport systems to deliver effectors to other cells and outer media. Generally, Bacterial secretion systems are protein complexes, present on the cell membranes of bacteria for secretion of substances. Specifically, they are the cellular devices used by pathogenic bacteria to secrete their virulence factors to invade the host cells. These multiprotein systems can be classified into different types based on their specific structure, composition and activity. Till now, nine different secretion pathways have been described – Type I to Type IX secretion systems (T1SS-T9SS). Most of the secretion systems have evolved from or co-evolved with different machineries such as motility, drug efflux or pilli assembly. In recent years, cryo-EM studies have a great role to understand these multiprotein complexes.

Mycobacterium tuberculosis (Mtb) is the causative agent of one of the deadliest infectious diseases known to man, tuberculosis. Mtb has a remarkable ability to persist inside the oxidatively hostile environment of human phagocytes encountering Reactive Oxygen Species, Reactive Nitrogen Species, low pH and nutrient starvation etc. This remarkable ability of Mtb made it a successful human pathogen. There are certain products generated from different metabolic pathways of Mtb helps it to combat hostile environment inside human. We are interested to know the mechanism as well as the structural details of proteins present in these kinds of metabolic pathway.

The iterative type I polyketide synthases (PKS) are multimodular mega-enzyme assembly lines responsible for the biosynthesis of many natural products (NP), which are used as the core structure of many pharmacologically diverse compounds like antibacterial, antifungal, anticancer and immunosuppressive compounds. Many of these NPs are either polyketide or nonribosomal peptide or hybrid backbone. Their biosynthetic pathways include one or more multifunctional PKS modules for the initiation, extension and modification steps leading to compounds such as leinamycin, erythromycin, tylosin and rapamycin. Bioengineering efforts to generate novel natural products, which in turn, can be used as valuable drugs, is a research topic of paramount importance for the past few decades. However, earlier efforts including domain modification and/or swapping to increase the productivity of natural products has failed due to lack of structural knowledge about the PKSs. Therefore, we can apply cryo-electron microscopy technique to study and understand the domain organization and interaction of PKS or NRPS-PKS ,which will also help us to apply new strategies to modify the domain organization to increase chemical diversity of these macromolecules.

We are also working on different bacterial cytosolic proteins, helical proteins etc. Developing new methods on cryo-electron microscopy is also our interest.

Publications

2020 1. Sameer Kumar Malladi, David Schreiberb, Ishika Pramanick, Malavika Abhineshababu Sridevi, Adi Goldenzweig, Somnath Dutta, Sarel Jacob Fleishman, Raghavan Varadarajana. One-step sequence and structure-guided optimization of HIV-1 envelope gp140. Current Research in Structural Biology, 2020.

2019 2. Eshan Ghosh, Hemlata Dwivedi, Mithu Baidya, Ashish Srivastava, Punita Kumari, Tomek Stepniewski, Hee Ryung Kim, Mi-Hye Lee, Jaana van Gastel, Madhu Chaturvedi, Debarati Roy, Shubhi Pandey, Jagannath Maharana, Ramon Guixà-González, Louis M Luttrell, Ka Young Chung, Somnath Dutta, Jana Selent, Arun K Shukla. Conformational Sensors and Domain Swapping Reveal Structural and Functional Differences Between β-Arrestin Isoforms. Cell Report, 2019.

3. Kumar S, Panda H, Makhdoomi MA, Mishra N, Safdari HA, Chawla H, Aggarwal H, Reddy ES, Lodha R, Kumar Kabra S, Chandele A, Dutta S, Luthra K. An HIV-1 Broadly Neutralizing Antibody from a Clade C-Infected Pediatric Elite Neutralizer Potently Neutralizes the Contemporaneous and Autologous Evolving Viruses. J Virol. 2019 3. Nidhi Kundu, Pratima Verma, Anil Kumar, Vinica Dhar, Somnath Dutta, Kausik Chattopadhyay. N-terminal region of Vibrio parahaemolyticus thermostable direct hemolysin regulates membrane-damaging action of the toxin. Biochemistry, 2019. 4. Koehl A, Hu H, Feng D, Sun B, Zhang Y, Robertson MJ, Chu M, Kobilka TS, Laeremans T, Steyaert J, Tarrasch J, Dutta S, Fonseca R, Weis WI, Mathiesen JM, Skiniotis G, Kobilka BK. Structural insights into the activation of metabotropic glutamate receptors. Nature. 2019 ;566(7742):79-84 5. Bhandari S, Biswas S, Chaudhary A, Dutta S, Suguna K. Dodecameric structure of a small heat shock protein from Mycobacterium marinum M. Proteins. 2019. 6. Madhurima Datta, Shashanka Arolia, Kapudeep Karmakara, Somnath Dutta, Dipshikha Chakravortty, Umesh Varshney. Development of mCherry tagged UdgX as a highly sensitive molecular probe for specific detection of uracils in DNA. Biochemical and biophysical research communication, 2019.

2018 6. Sadfari HA, Pandey S, Shukla AK, Dutta S. Illuminating GPCR Signaling by Cryo-EM. Trends Cell Biol. 2018 Volume 28, Issue 8, p591-594

Upto 2016: 1. De M, Oleskie AN, Ayyash M, Dutta S, Mancour L, Abazeed ME, Brace EJ, Skiniotis G, Fuller RS. The Vps13p-Cdc31p complex is directly required for TGN late endosome transport and TGN homotypic fusion. J Cell Biol. 2017, 216:425-439. 2. Yee A, Oleskie AN, Dosey AM, Kretz CA, Gildersleeve RD, Dutta S, Su M, Ginsburg D, Skiniotis G.Visualization of an N-terminal fragment of von Willebrand factor in complex with factor VIII. Blood. 2015, 126:939-42. 3.Dutta, S., Whicher, J. R., Hansen, D. A., Hale, W. A., Chemler, J. A., Congdon, G. R., Narayan, A. R.,

Håkansson, K., Sherman, D. H., Smith, J. L., and Skiniotis, G. Structure of a modular polyketide synthase. Nature. 2014, 510(7506):512-17. 4. Whicher, J. R., Dutta, S., Hansen, D. A., Hale, W. A., Chemler, J. A., Congdon, G. R., Narayan, A. R., Håkansson, K., Sherman, D. H., Smith, J. L., and Skiniotis, G. Structural rearrangements of a polyketide synthase module during its catalytic cycle. Nature. 2014, 510(7506):560-64. [*Equal contribution]. 5. Akey DL, Brown WC, Dutta S, Konwerski J, Jose J, Jurkiw TJ, DelProposto J, Ogata CM, Skiniotis G, Kuhn RJ, Smith JL. Flavivirus NS1 structures reveal surfaces for associations with membranes and the immune system. Science. 2014, 343(6173):881-85. 6. Dutta, S, Banerjee, KK, Ghosh AN. Cryo-electron microscopy reveals the membrane insertion mechanism of V. cholerae hemolysin. J Biomol Struct Dyn. 2013, 32(9):1434-42. 7. Lyon AM, Dutta S, Boguth CA, Skiniotis G, Tesmer JJ. Full-length Gα(q)-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain. Nat Struct Mol Biol. 2013, 20(3):355-62. 8. Mancour LV, Daghestani HN, Dutta S, Westfield GH, Schilling J, Oleskie AN, Herbstman JF, Chou SZ, Skiniotis G. Ligand-Induced Architecture of the Leptin Receptor Signaling Complex. Mol Cell. 2012, 48(4):655-61. 9. Westfield GH*, Rasmussen SG*, Su M*, Dutta S*, DeVree BT, Chung KY, Calinski D, Velez-Ruiz G, Oleskie AN, Pardon E, Chae PS, Liu T, Li S, Woods VL Jr, Steyaert J, Kobilka BK, Sunahara RK, Skiniotis G.Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex. Proc Natl Acad Sci. U S A. 2011, 108(38):16086-91. [* Equal contribution] 10. Gu L, Eisman EB, Dutta S, Franzmann TM, Walter S, Gerwick WH, Skiniotis G, Sherman DH. Tandem acyl carrier proteins in the curacin biosynthetic pathway promote consecutive multienzyme reactions with a synergistic effect. Angew Chem Int Ed Engl. 2011, 50(12):2795-98. 11. Dutta S, Mazumdar B, Banerjee KK, Ghosh AN. Three-dimensional structure of different functional forms of the Vibrio cholerae hemolysin oligomer: a cryo-electron microscopic study. J Bacteriol. 2010, 192(1):169-78.

Team members

PI : Dr. Somnath Dutta, Assistant Professor (from April 2016…..) Postdoc - University of Michigan, Ann Arbor (2010 -2016) Ph.D. - National Institute of Cholera and Enteric Diseases (From 2006-2010) Email ID: somnath@iisc.ac.in

Rupam Biswas (RA) I received my Bachelor's degree in Physics in 2008 and my Master's degree in Biophysics from Kalyani University in 2010. Soon after, I joined the Indian Institute of Technology (IIT) Kharagpur for a Ph.D. in Structural Biology program and received my Ph.D. in 2018. My doctoral research work at Dr. Amit Kumar Das’s laboratory entailed structural and biochemical insights into the enzymes involved in the type II pathway of bacterial fatty acid synthase. For my Postdoctoral research I joined Dr. Dutta’s laboratory in 2018 to study the

structural mechanisms of multidrug transporter complexes using cryo-electron microscopy. Email ID: rupambiswas@iisc.ac.in Anupam Roy (RA)

I was a graduate student at the Structural Biology and Bioinformatics Division of CSIR-Indian Institute of Chemical Biology, Kolkata. I had received my Ph.D from University of Calcutta, where my research as a graduate student had focused primarily on the structural aspects of different amyloids aggregates. Currently I’m working as a Research Associate at the Advanced Center for Cryo-

Electron Microscopy Facility in Dr. Somnath Dutta's Group, MBU-IISc and working in the field of Cryo-Electron Microscopy to understand structural overviews of bacterial pore forming toxins and viral phage proteins using SPA Cryo-EM. Email ID: anupamroy@iisc.ac.in Ishika Pramanick (PhD Student)

I did my Bachelor’s degree in Microbiology from Scottish Church College, Kolkata and post-graduation in Microbiology from University of Calcutta. I have joined Dr. Dutta’s lab in MBU since 2016 as a Graduate student. I am working on structural aspects of different macro-molecular biological samples using cryo-Electron Microscopy. Email ID: ishika@iisc.ac.in

Priyanka Garg (PhD Student)

I did my B.E. and M.E. in Biotechnology from Panjab University Chandigarh. I joined MBU as a graduate student in July 2017.I am currently working on structural and functional characterisation of Non-ribosomal peptide synthetase of leinamycin biosynthetic pathway. Email ID: priyanka2@iisc.ac.in

Nayanika Sengupta (PhD Student) I am a PhD student since July 2018, in the lab of Dr. Dutta. I pursued a BSc degree in Microbiology from St. Xavier’s College, Kolkata and went ahead to do an MSc in Biotechnology from University of Calcutta. Currently, I am invested in deciphering high resolution characteristics of Bacterial Secretion System proteins and Pore Forming Toxins. Email ID: nayanikas@iisc.ac.in

Suman Mishra (PhD Student) I joined Dr. Somnath Dutta's lab in July 2019 as a Ph.D. student. Prior to this, I completed my Bachelor of Technology (B.Tech) degree in Biotechnology from D.Y. Patil University, School of Biotechnology and Bioinformatics, Navi Mumbai, and did my six-months dissertation at CSIR- Center for Cellular and Molecular Biology (CCMB, Hyderabad) under Dr. K. Thangaraj. In this lab, my interests primarily include

structure elucidation of microbial proteins through cryo-EM. Email ID: sumanmishra@iisc.ac.in Surekha (PhD Student)

I did my bachelors in biotechnology from Rajalakshmi Engineering College, Chennai, Tamil Nadu. Followed by masters in medical biotechnology from Indian Institute of Technology, Hyderabad, Telangana. My interest in structural studies of macromolecular complexes has made me join Dr. Somnath’s lab in molecular biophysics department as a Ph.D student in August 2019. Email ID: surekhap@iisc.ac.in

Alakta Das (BS Student)

I am a final year undergraduate student at the Indian Institute of Science. I am working on Mycobacterial secretion system as a part of my final year project at the Dutta Lab. I am learning Cryo-EM, the cutting edge structural Biology tool and wish to pursue my future research career in the related field. Besides Science, I also take interest in writing poetry, playing ukulele and dancing Kathak. Email ID: alaktadas@iisc.ac.in

Gyana Prakash Mahapatra (Project Assistant)

I obtained Bachelor of Science (Hons) Biotechnology from Utkal University and Post Graduation in Marine Biotechnology from Annamalai University. I joined the lab in November 2019 to study the structural and functional mechanism of bacterial secretion system by employing state-of-the-art single particle Cryo-electron microscopy. Email ID: gyanam@iisc.ac.in

Chinmay KV (T12 TEM machine operator)

Email ID: k.v.chinmaya@gmail.com Anil Kumar (cryo-EM manager)

I completed my M.Sc in Biotechnology from Loyola college Chennai . Prior to this I completed my Bachelors in Biotechnology from Christ university. I am Maintaining cryo-EM facility at IISc, Bangalore. Email ID: anil.iiscmbu@gmail.com

Past Fellows 1. Alakta Das (May - June 2017), UG student of IISc – Summer training 2. Navyashree V (July-Sep 2017), Bangalore University – Summer training 3. Anuradha Choudhary (Project Fellow) May 2016-May 2018. (Now Ph.D. student at International Institute of Molecular and Cell Biology,Warsaw, Poland) 4. Abirlal Mukherjee (Project Fellow) 2018-2019. (Now Ph.D. student at Indian Institute of Technology, Roorkee) 5. Manish Sarkar (Project Fellow) 2016-2017 (Now Ph.D. Student at Bose Institute, Kolkata) 6. Suresh Kumar (Project Fellow) 7. Akansha Patel (M.Tech Final Year Project Student) 2016-2017 8. Preeti (Postdoc May 2017-May 2018) 9. Ayushi Shukla (Project Fellow) 10. Puja – Summer training 11. Haaris Ahsan Safdari (MS student 2017-2019)

Contacts Email ID: somnath@iisc.ac.in, sndutta.niced@gmail.com Phone number – (080) 2293-3453 Address - Molecular Biophysics Unit, Division of Biological Sciences, Indian Institute of Science, Bangalore - 560012, INDIA

Instruments

1.Talos Arctica 200 kV cryo-TEM 2. Vitrobot 3. 120 kV Biotwin TEM 4. Carbon Coater and Glow Discharger 5. Highly sophisticated data processing server and GPU work workstations 6. Protein purification Equipments

Course work 1. Electron microscopy and 3D image processing for Life sciences. (MB 212 JAN 2:0)