skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Functional plasticity of antibacterial EndoU toxins

Abstract

Bacteria use several different secretion systems to deliver toxic EndoU ribonucleases into neighboring cells. Here, we present the first structure of a prokaryotic EndoU toxin in complex with its cognate immunity protein. The contact-dependent growth inhibition toxin CdiA-CTSTECO31 from Escherichia coli STEC_O31 adopts the eukaryotic EndoU fold and shares greatest structural homology with the nuclease domain of coronavirus Nsp15. The toxin contains a canonical His-His-Lys catalytic triad in the same arrangement as eukaryotic EndoU domains, but lacks the uridylate-specific ribonuclease activity that characterizes the superfamily. Comparative sequence analysis indicates that bacterial EndoU domains segregate into at least three major clades based on structural variations in the N-terminal subdomain. Representative EndoU nucleases from clades I and II degrade tRNA molecules with little specificity. In contrast, CdiA-CTSTECO31 and other clade III toxins are specific anticodon nucleases that cleave tRNAGlu between nucleotides C37 and m2A38. In conclusion, these findings suggest that the EndoU fold is a versatile scaffold for the evolution of novel substrate specificities. Such functional plasticity may account for the widespread use of EndoU effectors by diverse inter-bacterial toxin delivery systems.

Authors:
 [1];  [2];  [2];  [1];  [1];  [2];  [2];  [3];  [2];  [4];  [5];  [2]
  1. Argonne National Lab. (ANL), Argonne, IL (United States)
  2. Univ. of California, Santa Barbara, CA (United States)
  3. Uppsala Univ., Uppsala (Sweden)
  4. Univ. of California, Irvine, CA (United States)
  5. Argonne National Lab. (ANL), Argonne, IL (United States); Univ. of Chicago, Chicago, IL (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1491848
Alternate Identifier(s):
OSTI ID: 1464280
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
Molecular microbiology
Additional Journal Information:
Journal Volume: 109; Journal Issue: 4; Journal ID: ISSN 0950-382X
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Michalska, Karolina, Nhan, Dinh Quan, Willett, Julia L. E., Stols, Lucy M., Eschenfeldt, William H., Jones, Allison M., Nguyen, Josephine Y., Koskiniemi, Sanna, Low, David A., Goulding, Celia W., Joachimiak, Andrzej, and Hayes, Christopher S. Functional plasticity of antibacterial EndoU toxins. United States: N. p., 2018. Web. doi:10.1111/mmi.14007.
Michalska, Karolina, Nhan, Dinh Quan, Willett, Julia L. E., Stols, Lucy M., Eschenfeldt, William H., Jones, Allison M., Nguyen, Josephine Y., Koskiniemi, Sanna, Low, David A., Goulding, Celia W., Joachimiak, Andrzej, & Hayes, Christopher S. Functional plasticity of antibacterial EndoU toxins. United States. doi:https://doi.org/10.1111/mmi.14007
Michalska, Karolina, Nhan, Dinh Quan, Willett, Julia L. E., Stols, Lucy M., Eschenfeldt, William H., Jones, Allison M., Nguyen, Josephine Y., Koskiniemi, Sanna, Low, David A., Goulding, Celia W., Joachimiak, Andrzej, and Hayes, Christopher S. Wed . "Functional plasticity of antibacterial EndoU toxins". United States. doi:https://doi.org/10.1111/mmi.14007. https://www.osti.gov/servlets/purl/1491848.
@article{osti_1491848,
title = {Functional plasticity of antibacterial EndoU toxins},
author = {Michalska, Karolina and Nhan, Dinh Quan and Willett, Julia L. E. and Stols, Lucy M. and Eschenfeldt, William H. and Jones, Allison M. and Nguyen, Josephine Y. and Koskiniemi, Sanna and Low, David A. and Goulding, Celia W. and Joachimiak, Andrzej and Hayes, Christopher S.},
abstractNote = {Bacteria use several different secretion systems to deliver toxic EndoU ribonucleases into neighboring cells. Here, we present the first structure of a prokaryotic EndoU toxin in complex with its cognate immunity protein. The contact-dependent growth inhibition toxin CdiA-CTSTECO31 from Escherichia coli STEC_O31 adopts the eukaryotic EndoU fold and shares greatest structural homology with the nuclease domain of coronavirus Nsp15. The toxin contains a canonical His-His-Lys catalytic triad in the same arrangement as eukaryotic EndoU domains, but lacks the uridylate-specific ribonuclease activity that characterizes the superfamily. Comparative sequence analysis indicates that bacterial EndoU domains segregate into at least three major clades based on structural variations in the N-terminal subdomain. Representative EndoU nucleases from clades I and II degrade tRNA molecules with little specificity. In contrast, CdiA-CTSTECO31 and other clade III toxins are specific anticodon nucleases that cleave tRNAGlu between nucleotides C37 and m2A38. In conclusion, these findings suggest that the EndoU fold is a versatile scaffold for the evolution of novel substrate specificities. Such functional plasticity may account for the widespread use of EndoU effectors by diverse inter-bacterial toxin delivery systems.},
doi = {10.1111/mmi.14007},
journal = {Molecular microbiology},
number = 4,
volume = 109,
place = {United States},
year = {2018},
month = {6}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 4 works
Citation information provided by
Web of Science

Figures / Tables:

Table 1 Table 1: Data processing and refinement statistics

Save / Share:

Works referenced in this record:

The active site and mechanism of action of bovine pancreatic ribonuclease. 7. The catalytic mechanism
journal, October 1962

  • Findlay, D.; Herries, Dg; Mathias, Ap
  • Biochemical Journal, Vol. 85, Issue 1
  • DOI: 10.1042/bj0850152

MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes
journal, July 2004

  • Davis, I. W.; Murray, L. W.; Richardson, J. S.
  • Nucleic Acids Research, Vol. 32, Issue Web Server
  • DOI: 10.1093/nar/gkh398

New LIC vectors for production of proteins from genes containing rare codons
journal, September 2013

  • Eschenfeldt, William H.; Makowska-Grzyska, Magdalena; Stols, Lucy
  • Journal of Structural and Functional Genomics, Vol. 14, Issue 4
  • DOI: 10.1007/s10969-013-9163-9

Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage
journal, August 2003


Ribosomally encoded antibacterial proteins and peptides from Pseudomonas
journal, July 2014


CdiA Effectors from Uropathogenic Escherichia coli Use Heterotrimeric Osmoporins as Receptors to Recognize Target Bacteria
journal, October 2016


The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria
journal, October 2016


CdiA from Enterobacter cloacae Delivers a Toxic Ribosomal RNase into Target Bacteria
journal, May 2014


Refinement of Macromolecular Structures by the Maximum-Likelihood Method
journal, May 1997

  • Murshudov, G. N.; Vagin, A. A.; Dodson, E. J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 53, Issue 3
  • DOI: 10.1107/S0907444996012255

Biochemical and Genetic Analyses of Murine Hepatitis Virus Nsp15 Endoribonuclease
journal, September 2007


Overview of the CCP 4 suite and current developments
journal, March 2011

  • Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444910045749

Biochemical Characterization of Arterivirus Nonstructural Protein 11 Reveals the Nidovirus-Wide Conservation of a Replicative Endoribonuclease
journal, March 2009

  • Nedialkova, D. D.; Ulferts, R.; van den Born, E.
  • Journal of Virology, Vol. 83, Issue 11
  • DOI: 10.1128/JVI.00261-09

Cleavage of the A Site mRNA Codon during Ribosome Pausing Provides a Mechanism for Translational Quality Control
journal, October 2003


Dali server: conservation mapping in 3D
journal, May 2010

  • Holm, Liisa; Rosenstr�m, P�ivi
  • Nucleic Acids Research, Vol. 38, Issue suppl_2
  • DOI: 10.1093/nar/gkq366

The structure of a contact-dependent growth-inhibition (CDI) immunity protein from Neisseria meningitidis MC58
journal, May 2015

  • Tan, Kemin; Johnson, Parker M.; Stols, Lucy
  • Acta Crystallographica Section F Structural Biology Communications, Vol. 71, Issue 6
  • DOI: 10.1107/S2053230X15006585

Contact-dependent growth inhibition requires the essential outer membrane protein BamA (YaeT) as the receptor and the inner membrane transport protein AcrB
journal, October 2008


Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors
journal, February 2017

  • Jones, Allison M.; Garza-Sánchez, Fernando; So, Jaime
  • Proceedings of the National Academy of Sciences, Vol. 114, Issue 10
  • DOI: 10.1073/pnas.1619273114

Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways
journal, August 2015

  • Willett, Julia L. E.; Gucinski, Grant C.; Fatherree, Jackson P.
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 36
  • DOI: 10.1073/pnas.1512124112

Engineering Controllable Protein Degradation
journal, June 2006


Purification, Cloning, and Characterization of XendoU, a Novel Endoribonuclease Involved in Processing of Intron-encoded Small Nucleolar RNAs in Xenopus laevis
journal, February 2003

  • Laneve, Pietro; Altieri, Fabio; Fiori, Micol E.
  • Journal of Biological Chemistry, Vol. 278, Issue 15
  • DOI: 10.1074/jbc.M211937200

Selection of Orphan Rhs Toxin Expression in Evolved Salmonella enterica Serovar Typhimurium
journal, March 2014


Unraveling the essential role of CysK in CDI toxin activation
journal, August 2016

  • Johnson, Parker M.; Beck, Christina M.; Morse, Robert P.
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 35
  • DOI: 10.1073/pnas.1607112113

Contact-Dependent Inhibition of Growth in Escherichia coli
journal, August 2005


Structural Biology of the Arterivirus nsp11 Endoribonucleases
journal, October 2016

  • Zhang, Manfeng; Li, Xiaorong; Deng, Zengqin
  • Journal of Virology, Vol. 91, Issue 1
  • DOI: 10.1128/JVI.01309-16

Bovine Pancreatic Ribonuclease: Fifty Years of the First Enzymatic Reaction Mechanism
journal, September 2011

  • Cuchillo, Claudi M.; Nogués, M. Victòria; Raines, Ronald T.
  • Biochemistry, Vol. 50, Issue 37
  • DOI: 10.1021/bi201075b

New Antiviral Target Revealed by the Hexameric Structure of Mouse Hepatitis Virus Nonstructural Protein nsp15
journal, July 2006


A New Family of Secreted Toxins in Pathogenic Neisseria Species
journal, January 2015


Genetically distinct pathways guide effector export through the type VI secretion system: Effector export pathways of type VI secretion
journal, March 2014

  • Whitney, John C.; Beck, Christina M.; Goo, Young Ah
  • Molecular Microbiology, Vol. 92, Issue 3
  • DOI: 10.1111/mmi.12571

Inference of Macromolecular Assemblies from Crystalline State
journal, September 2007


A statistic for local intensity differences: robustness to anisotropy and pseudo-centering and utility for detecting twinning
journal, June 2003

  • Padilla, Jennifer E.; Yeates, Todd O.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 59, Issue 7
  • DOI: 10.1107/S0907444903007947

UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

  • Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
  • Journal of Computational Chemistry, Vol. 25, Issue 13
  • DOI: 10.1002/jcc.20084

In vitro study of processing of the intron-encoded U16 small nucleolar RNA in Xenopus laevis.
journal, May 1994

  • Caffarelli, E.; Arese, M.; Santoro, B.
  • Molecular and Cellular Biology, Vol. 14, Issue 5
  • DOI: 10.1128/MCB.14.5.2966

Mutational Analysis of the SARS Virus Nsp15 Endoribonuclease: Identification of Residues Affecting Hexamer Formation
journal, November 2005

  • Guarino, Linda A.; Bhardwaj, Kanchan; Dong, Wen
  • Journal of Molecular Biology, Vol. 353, Issue 5
  • DOI: 10.1016/j.jmb.2005.09.007

Domain exchange at the 3’ end of the gene encoding the fratricide meningococcal two-partner secretion protein A
journal, January 2013


Can't you hear me knocking: contact-dependent competition and cooperation in bacteria
journal, April 2017

  • Jones, Allison M.; Low, David A.; Hayes, Christopher S.
  • Emerging Topics in Life Sciences, Vol. 1, Issue 1
  • DOI: 10.1042/ETLS20160019

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins
journal, March 2017

  • García-Bayona, Leonor; Guo, Monica S.; Laub, Michael T.
  • eLife, Vol. 6
  • DOI: 10.7554/eLife.24869

The calcium-dependent ribonuclease XendoU promotes ER network formation through local RNA degradation
journal, October 2014

  • Schwarz, Dianne S.; Blower, Michael D.
  • The Journal of Cell Biology, Vol. 207, Issue 1
  • DOI: 10.1083/jcb.201406037

The Buccaneer software for automated model building. 1. Tracing protein chains
journal, August 2006


Structural and Functional Analyses of the Severe Acute Respiratory Syndrome Coronavirus Endoribonuclease Nsp15
journal, November 2007

  • Bhardwaj, Kanchan; Palaninathan, Satheesh; Alcantara, Joanna Maria Ortiz
  • Journal of Biological Chemistry, Vol. 283, Issue 6
  • DOI: 10.1074/jbc.M708375200

Colicin Biology
journal, March 2007

  • Cascales, E.; Buchanan, S. K.; Duche, D.
  • Microbiology and Molecular Biology Reviews, Vol. 71, Issue 1
  • DOI: 10.1128/MMBR.00036-06

Mechanism of Action of Bovine Pancreatic Ribonuclease
journal, July 1966

  • Deavin, A.; Mathias, A. P.; Rabin, B. R.
  • Nature, Vol. 211, Issue 5046
  • DOI: 10.1038/211252a0

A short history of SHELX
journal, December 2007

  • Sheldrick, George M.
  • Acta Crystallographica Section A Foundations of Crystallography, Vol. 64, Issue 1, p. 112-122
  • DOI: 10.1107/S0108767307043930

Structure of a novel antibacterial toxin that exploits elongation factor Tu to cleave specific transfer RNAs
journal, August 2017

  • Michalska, Karolina; Gucinski, Grant C.; Garza-Sánchez, Fernando
  • Nucleic Acids Research, Vol. 45, Issue 17
  • DOI: 10.1093/nar/gkx700

HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes
journal, July 2006

  • Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek
  • Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8
  • DOI: 10.1107/S0907444906019949

Type VI secretion system effectors: poisons with a purpose
journal, January 2014

  • Russell, Alistair B.; Peterson, S. Brook; Mougous, Joseph D.
  • Nature Reviews Microbiology, Vol. 12, Issue 2
  • DOI: 10.1038/nrmicro3185

Functional Characterization of XendoU, the Endoribonuclease Involved in Small Nucleolar RNA Biosynthesis
journal, March 2005

  • Gioia, Ubaldo; Laneve, Pietro; Dlakić, Mensur
  • Journal of Biological Chemistry, Vol. 280, Issue 19
  • DOI: 10.1074/jbc.M501160200

The Vibrio cholerae type VI secretion system employs diverse effector modules for intraspecific competition
journal, April 2014

  • Unterweger, Daniel; Miyata, Sarah T.; Bachmann, Verena
  • Nature Communications, Vol. 5, Issue 1
  • DOI: 10.1038/ncomms4549

A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria
journal, November 2010

  • Aoki, Stephanie K.; Diner, Elie J.; de Roodenbeke, Claire t’Kint
  • Nature, Vol. 468, Issue 7322
  • DOI: 10.1038/nature09490

Coot model-building tools for molecular graphics
journal, November 2004

  • Emsley, Paul; Cowtan, Kevin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
  • DOI: 10.1107/S0907444904019158

Deciphering key features in protein structures with the new ENDscript server
journal, April 2014

  • Robert, Xavier; Gouet, Patrice
  • Nucleic Acids Research, Vol. 42, Issue W1
  • DOI: 10.1093/nar/gku316

A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems
journal, February 2011

  • Zhang, Dapeng; Iyer, Lakshminarayan M.; Aravind, L.
  • Nucleic Acids Research, Vol. 39, Issue 11
  • DOI: 10.1093/nar/gkr036

Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages
journal, May 2017

  • Deng, Xufang; Hackbart, Matthew; Mettelman, Robert C.
  • Proceedings of the National Academy of Sciences, Vol. 114, Issue 21
  • DOI: 10.1073/pnas.1618310114

A View to a Kill: The Bacterial Type VI Secretion System
journal, January 2014


Bacterial Quorum-Sensing Network Architectures
journal, December 2009


The toxin/immunity network of Burkholderia pseudomallei contact-dependent growth inhibition (CDI) systems: Burkholderia pseudomallei CDI systems
journal, April 2012


Prolyl-tRNA Pro in the A-site of SecM-arrested Ribosomes Inhibits the Recruitment of Transfer-messenger RNA
journal, September 2006

  • Garza-Sánchez, Fernando; Janssen, Brian D.; Hayes, Christopher S.
  • Journal of Biological Chemistry, Vol. 281, Issue 45
  • DOI: 10.1074/jbc.M608052200

A Type VI Secretion System of Pseudomonas aeruginosa Targets a Toxin to Bacteria
journal, January 2010


Bacterial killing via a type IV secretion system
journal, March 2015

  • Souza, Diorge P.; Oka, Gabriel U.; Alvarez-Martinez, Cristina E.
  • Nature Communications, Vol. 6, Issue 1
  • DOI: 10.1038/ncomms7453

A novel family of toxin/antitoxin proteins in Bacillus species
journal, December 2011


CDI Systems Are Stably Maintained by a Cell-Contact Mediated Surveillance Mechanism
journal, June 2016


Cleavable C-terminal His-tag vectors for structure determination
journal, March 2010

  • Eschenfeldt, William H.; Maltseva, Natalia; Stols, Lucy
  • Journal of Structural and Functional Genomics, Vol. 11, Issue 1
  • DOI: 10.1007/s10969-010-9082-y

A broadly distributed toxin family mediates contact-dependent antagonism between gram-positive bacteria
journal, July 2017


On the treatment of negative intensity observations
journal, July 1978


Contact-Dependent Growth Inhibition (CDI) and CdiB/CdiA Two-Partner Secretion Proteins
journal, November 2015

  • Willett, Julia L. E.; Ruhe, Zachary C.; Goulding, Celia W.
  • Journal of Molecular Biology, Vol. 427, Issue 23
  • DOI: 10.1016/j.jmb.2015.09.010

Sibling Rivalry in Myxococcus xanthus Is Mediated by Kin Recognition and a Polyploid Prophage
journal, January 2016

  • Dey, Arup; Vassallo, Christopher N.; Conklin, Austin C.
  • Journal of Bacteriology, Vol. 198, Issue 6
  • DOI: 10.1128/JB.00964-15

EssD, a Nuclease Effector of the Staphylococcus aureus ESS Pathway
journal, October 2016

  • Ohr, Ryan Jay; Anderson, Mark; Shi, Miaomiao
  • Journal of Bacteriology, Vol. 199, Issue 1
  • DOI: 10.1128/JB.00528-16

Type VI secretion requires a dynamic contractile phage tail-like structure
journal, February 2012

  • Basler, M.; Pilhofer, M.; Henderson, G. P.
  • Nature, Vol. 483, Issue 7388
  • DOI: 10.1038/nature10846

Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI)
journal, February 2012


The structure of the endoribonuclease XendoU: From small nucleolar RNA processing to severe acute respiratory syndrome coronavirus replication
journal, August 2006

  • Renzi, F.; Caffarelli, E.; Laneve, P.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 33
  • DOI: 10.1073/pnas.0602426103

The Vibrio cholerae type VI secretion system displays antimicrobial properties
journal, October 2010

  • MacIntyre, D. L.; Miyata, S. T.; Kitaoka, M.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 45
  • DOI: 10.1073/pnas.1012931107

Major genetic marker of nidoviruses encodes a replicative endoribonuclease
journal, August 2004

  • Ivanov, K. A.; Hertzig, T.; Rozanov, M.
  • Proceedings of the National Academy of Sciences, Vol. 101, Issue 34
  • DOI: 10.1073/pnas.0403127101

Crystal structure and mechanistic determinants of SARS coronavirus nonstructural protein 15 define an endoribonuclease family
journal, August 2006

  • Ricagno, S.; Egloff, M. -P.; Ulferts, R.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 32
  • DOI: 10.1073/pnas.0601708103

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication
journal, February 2017


A Novel Mn++-Dependent Ribonuclease That Functions in U16 SnoRNA Processing inX.Laevis.
journal, April 1997

  • Caffarelli, Elisa; Maggi, Laura; Fatica, Alessandro
  • Biochemical and Biophysical Research Communications, Vol. 233, Issue 2
  • DOI: 10.1006/bbrc.1997.6487

Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems
journal, August 2011


Rhs proteins from diverse bacteria mediate intercellular competition
journal, April 2013

  • Koskiniemi, S.; Lamoureux, J. G.; Nikolakakis, K. C.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 17
  • DOI: 10.1073/pnas.1300627110

A Dimerization-Dependent Mechanism Drives the Endoribonuclease Function of Porcine Reproductive and Respiratory Syndrome Virus nsp11
journal, February 2016

  • Shi, Yuejun; Li, Youwen; Lei, Yingying
  • Journal of Virology, Vol. 90, Issue 9
  • DOI: 10.1128/JVI.03065-15

The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor
journal, October 2004


Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems
journal, December 2012

  • Morse, R. P.; Nikolakakis, K. C.; Willett, J. L. E.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 52
  • DOI: 10.1073/pnas.1216238110

    Works referencing / citing this record:

    Target highlights in CASP13: Experimental target structures through the eyes of their authors
    journal, August 2019

    • Lepore, Rosalba; Kryshtafovych, Andriy; Alahuhta, Markus
    • Proteins: Structure, Function, and Bioinformatics, Vol. 87, Issue 12
    • DOI: 10.1002/prot.25805