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Accurate size-based protein localization from cryo-ET tomograms

Weisheng Jin^a, Ye Zhou^a, Alberto Bartesaghi ^a,b,c,*

^a Department of Computer Science, Duke University

^b Department of Biochemistry, Duke University School of Medicine

^c Department of Electrical and Computer Engineering, Pratt School of Engineering, Duke University

_{Fig. 4. Maps from particles obtained using our size-based approach and crYOLO. Reconstructions obtained by sub-tomogram averaging from particles produced using our size-based particle picking approach and crYOLO for tomograms from EMPIAR-10064 and EMPIAR-10499.}

Abstract

Cryo-electron tomography (cryo-ET) combined with sub-tomogram averaging (STA) allows the determination of protein structures imaged within the native context of the cell at near-atomic resolution. Particle picking is an essential step in the cryo-ET/STA image analysis pipeline that consists in locating the position of proteins within
crowded cellular tomograms so that they can be aligned and averaged in 3D to improve resolution. While extensive work in 2D particle picking has been done in the context of single-particle cryo-EM, comparatively fewer strategies have been proposed to pick particles from 3D tomograms, in part due to the challenges asso-
ciated with working with noisy 3D volumes affected by the missing wedge. While strategies based on 3D template-matching and deep learning are commonly used, these methods are computationally expensive and require either an external template or manual labelling which can bias the results and limit their applicability.
Here, we propose a size-based method to pick particles from tomograms that is fast, accurate, and does not require external templates or user provided labels. We compare the performance of our approach against a commonly used algorithm based on deep learning, crYOLO, and show that our method: i) has higher detection
accuracy, ii) does not require user input for labeling or time-consuming training, and iii) runs efficiently on non-specialized CPU hardware. We demonstrate the effectiveness of our approach by automatically detecting par-ticles from tomograms representing different types of samples and using these particles to determine the high-resolution structures of ribosomes imaged in vitro and in situ.

Advances in cryo-ET data processing: meeting the demands of visual proteomics

Abigail J. I. Watson¹ and Alberto Bartesaghi ^1,2,3

¹ Department of Biochemistry, Duke University School of Medicine
² Department of Computer Science, Duke University
³ Department of Electrical and Computer Engineering, Duke University

_{Published figure: Overview of strategies for particle picking from cryo-ET tomograms. Reconstructed tomograms in 3D are used as input to the particle picking method of choice.}

Abstract
Cryogenic electron tomography (cryo-ET), a method that enables the viewing of biomolecules in near-native environments at high resolution, is rising in accessibility and applicability. Over the past several years, once slow sample preparation and data collection procedures have seen innovations which enable rapid collection of the large datasets required for attaining high resolution structures. Increased data availability has provided a driving force for exciting improvements in cryo-ET data processing  methodologies throughout the entire processing pipeline and the development of  accessible graphical user interfaces (GUIs) that enable individuals inexperienced in computational fields to convert raw tilt series into 3D structures. These advances in data processing are enabling cryo-ET to attain higher resolution and extending its applicability to more complex samples.

Microsecond dynamics control the HIV-1 envelope conformation

Ashley L. Bennett¹, R.J. Edwards¹, Irina Kosheleva², Carrie Saunders¹, Yishak Bililign¹,
Ashliegh Williams¹, Katayoun Manosouri¹, Kevin O. Saunders^1,3, Barton F. Haynes ^1,4,5,
Priyamvada Acharya^1,3,5, Rory Henderson^1,6
 

¹Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710, USA.
²BioCARS, Center for Advanced Radiation Sources, The University of Chicago, 9700 South
  Cass Ave, Bld 434B, Lemont, IL 60439, USA.
³Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
⁴Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA.
⁵Department of Biochemistry, Duke University, Durham, NC 27710, USA
⁶Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA

Vaccine induction of CD4-mimicking broadly neutralizing antibody precursors in macaques

Kevin O. Saunders^1,2,3,4,14, James Counts^1,5, Victoria Stalls^1,5 , Robert Edwards^1,5, Kartik⁵, Manne^1,5, Xiaozhi Lu^1,5, Bhishem Thakur^1,5, Katayoun Mansouri^1,5 , Yue Chen^1,5 , Rob Parks^1,5, Maggie Barr^1,5, Laura Sutherland^1,5, Joena Bal^1,5, Nicholas Havill^1,6, Haiyan Chen^1,5, Emily Machiele^1,5, Nolan Jamieson^1,5, Bhavna Hora^1,5, Megan Kopp^1,5, Katarzyna Janowska^1,5, Kara Anasti^1,5, Chuancang Jiang^1,5, Sravani Venkatayogi ^1,5, Amanda Eaton^1,5, Rory Henderson^1,5, Christopher Barbosa⁷, S. Munir Alam^1,5, Sampa Santra⁸, Drew Weissman^9,10, M. Anthony Moody^1,11 , Derek W. Cain^1,5, Ying Tam⁷, Mark Lewis¹², Wilton B. Williams^1,2,3 , Kevin Wiehe^1,5 , David Montefiori^1,2, Priyamvada Acharya ^1,2,13 , Barton F. Haynes^1,3,4,5.

¹   Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710,USA.
²   Department of Surgery, Duke University School of Medicine, Durham, NC 27710, USA.
³   Department of Immunology, Duke University School of Medicine, Durham, NC 27710, USA.
⁴   Department of Molecular Genetics and Microbiology, Duke University School of Medicine,
⁵   Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA.
⁶   Department of Biology, Davidson College, Davidson, NC 28035, USA
⁷   Acuitas LLC, Vancouver, BC
⁸   Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
⁹   Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA.
¹⁰ Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
¹¹ Department of Pediatrics, Duke University School of Medicine, Durham, NC 27710, USA.
¹² BIOQUAL, Rockville, MD 20850, USA.
¹³ Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA.
¹⁴ Lead contact

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Researchers in the Duke Center for HIV Structural Biology and MIT have developed and validated a novel, high-throughput approach for structure determination by single-particle cryo-electron tomography using in vitro and cellular datasets, demonstrating its effectiveness at achieving high-resolution and revealing conformational heterogeneity in situ. The framework is made available through an intuitive and easy-to-use computer application, nextPYP.

Check out the full article by Dr. Hsuan-Fu Liu et al, in Nature Methods!

Modified Figure 1. V3-glycan targeting DH270 bnAb phylogeny.

The inferred DH270 clone phylogenetic tree depicting cryo-EM maps determined for each clonal member Fab bound to the CH848 d949 trimer (white). The clonal member names are colored according to the Fab color of each map.

Check out the full article by Rory Henderson et al. in Nature Communications!

Modified Supplemental Figure 4: Fusion peptide comparison.

Comparison of fusion peptide location in a selected group of viral fusion proteins. In the top portion of each panel, fusion proteins are shown in cartoon representation in gray, with the fusion peptide as orange spheres, and in the bottom portion, fusion peptides are shown as cartoon representation with side chains as sticks, orange, with the surface of the rest of the fusion protein colored based on protomer.

Check out the full article by Aaron May et al. in the Journal of Virology!

Vaccine Induction in Humans of Polyclonal HIV-1 Heterologous Neutralizing Antibodies

Wilton B. Williams^1,2,5,*&, S. Munir Alam^1,4,*&, Gilad Ofek^3,15,&, Nathaniel Erdmann^9,&, David
Montefiori^1,2,&, Michael Seaman¹², Kshitij Wagh¹⁴, Bette Korber¹⁴, Robert J. Edwards^1,4,
Katayoun Mansouri¹, Amanda Eaton², Derek W. Cain^1,4, Robert Parks¹, Maggie Barr¹, Mitchell
Martin¹, Jongln Hwang¹, Andrew Foulger¹, Kara Anasti¹, Salam Sammour¹, Xiao Huang¹, Jared
Lindenberger¹, Katarzyna Janowska¹, Aurelie Niyongabo³, Benjamin M. Janus^3,15, Anagh
Astavans³, Christopher Fox¹³, Ipsita Mohanty¹, Tyler Evangelous¹, Madison Berry¹, Helene
Kirshner¹, Kevin Saunders^1,2,5,6, Kevin Wiehe^1,4, Kristen Cohen⁷, M. Juliana McElrath⁷,
Lawrence Corey⁷, Priyamvada Acharya^1,2,10, Stephen R. Walsh^8,*, Lindsey R. Baden^8,* and
Barton F. Haynes^1,4,5,11,*.

¹Duke Human Vaccine Institute, Duke School of Medicine, Durham, NC 27710
²Department of Surgery, Duke School of Medicine, Durham, NC 27710
³Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742
⁴Department of Medicine, Duke School of Medicine, Durham, NC 27710
⁵Department of Immunology, Duke School of Medicine, Durham, NC 27710
⁶Department of Molecular Genetics and Microbiology, Duke School of Medicine, Durham, NC 27710
⁷Fred Hutchinson Cancer Research Center, Seattle WA 98109
⁸Brigham and Women’s Hospital, Harvard Medical School, Boston MA 02115
⁹University of Alabama Medical Center, Birmingham AL 35223
¹⁰Department of Biochemistry, Duke School of Medicine, Durham, NC 27710
¹¹Duke Global Health Institute, Duke School of Medicine, Durham, NC 27710
¹²Beth Israel Deaconess Medical Center, Harvard Medical School, Boston MA 02215
¹³Access to Access to Advanced Health Institute, Seattle WA, 98102
¹⁴Los Alamos National Laboratory, Los Alamos NM, 87545
¹⁵Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville MD 20850
^&These authors contributed equally to this work.
*Corresponding Authors: wilton.williams@duke.edu; munir.alam@duke.edu;
barton.haynes@duke.edu; lbaden@bwh.harvard.edu; SWALSH@bwh.harvard.edu.

Dynamics and Activation of Membrane-Bound B Cell Receptor Assembly

Hung N. Do¹, Mingfei Zhao^1,2, S. Munir Alam³, S. Gnankaran¹

¹ Los Alamos National Laboratory, ² University of Alabama (new appointment), ³Duke University, Duke Human Vaccine Institute

B-cell receptor complexes (BCR) are expressed on the surface of a B-cell and are the critical regulators of adaptive immune response. Even though the relevance of antibodies has been known for almost a hundred years, the antigen-dependent activation of antibody-producing B-cells has remained elusive. Several models have been proposed for BCR activation, including cross-linking, conformation-induced oligomerization, and dissociation activation models. Recently, the first cryo-EM structure of the human B-cell antigen receptor of the IgM isotype was published. Given the new asymmetric BCR complex, we have carried out extensive molecular dynamics simulations to probe the conformational changes upon antigen binding and the influence of the membrane. We identified two critical dynamical events that could be associated with antigen-dependent activation of BCR. First, antigen binding caused increased flexibility in regions distal to the antigen binding site. Second, this increased flexibility led to the rearrangement of helices in transmembrane helices, including the relative interaction of Igα/Igβ, which has been responsible for intracellular signaling. Further, these transmembrane rearrangements led to changes in localized lipid composition. Even though the simulations considered only a single BCR complex, our work indirectly supports the dissociation activation model.

Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted/ founder (TF) sensitivity to broadly neutralizing antibodies

Durgadevi Parthasarathy^1,10, Karunakar Reddy Pothula^3,10, Kim-Marie A. Dam², Sneha Ratnapriya¹, Héctor Cervera Benet¹, Ruth Parsons³, Xiao Huang³ , Salam Sammour³, Katarzyna
Janowska³, Miranda Harris¹, Samuel Sacco^2,11, Joseph Sodroski⁴, Michael D. Bridges^5,6, Wayne L. Hubbell^5,6, Priyamvada Acharya^3,7 and Alon Herschhorn^1,8,9 *

¹ Division of Infectious Diseases and International Medicine, Department of Medicine, University of Minnesota, Minneapolis, MN, USA
² Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
³ Duke Human Vaccine Institute, Durham, NC, USA
⁴ Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA; Department of  Microbiology, Harvard Medical School, Boston, MA, USA
⁵ Jules Stein Eye Institute, University of California, Los Angeles, CA, USA
⁶ Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA
⁷ Department of Surgery, and Department of Biochemistry, Duke University, Durham, NC, USA
⁸ Institute for Molecular Virology, University of Minnesota, Minneapolis, MN, USA
⁹ Microbiology, Immunology, and Cancer Biology Graduate Program; The College of Veterinary Medicine
  Graduate Program; and the Molecular Pharmacology and Therapeutics Graduate Program, University of
  Minnesota, Minneapolis, MN, USA
¹⁰ These authors contributed equally:  Durgadevi Parthasarathy, Karunakar Reddy Pothula.
¹¹ Present address: Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa
Cruz, CA, USA

*Correspondence:
Alon Herschhornl: aherschh@umn.edu

An intact amber-free HIV-1 system for in-virus protein bioorthogonal click labeling that delineates envelope conformational dynamics

Yuanyun Ao¹, Jonathan R. Grover², Yang Han¹, Guohua Zhong¹, Wenyi Qin¹, Dibya
Ghimire¹, Md. Anzarul Haque¹, Rajanya Bhattacharjee^3,4, Baoshan Zhang⁵, James
Arthos⁶, Edward A. Lemke^3,4, Peter D. Kwong⁵, *Maolin Lu¹

¹Department of Cellular and Molecular Biology, School of Medicine, University of Texas at Tyler Health Science Center, Tyler, Texas, USA
²Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA
³Biocentre, Departments of Biology and Chemistry, Johannes Gutenberg–University Mainz, Hanns-Dieter-Hüsch-Weg 17, Mainz, Germany
⁴Institute of Molecular Biology, Ackermannweg 4, Mainz, Germany
⁵Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
⁶Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
*Correspondence should be addressed to M.L.: maolin.lu@uthct.edu