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Molecular Biology 2563
Huan-Xiang Zhou
Jan-Hendrik Spille
Priya R. Banerjee Editors
Phase-Separated
Biomolecular
Condensates
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
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School of Life and Medical Sciences
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Phase-Separated Biomolecular
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Methods and Protocols
Edited by
Huan-Xiang Zhou
Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA
Jan-Hendrik Spille
Department of Physics, University of Illinois at Chicago, Chicago, IL, USA
Priya R. Banerjee
Department of Physics, University at Buffalo, State University, Buffalo, NY, USA
Editors
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Department of Chemistry Department of Physics
University of Illinois at Chicago University of Illinois at Chicago
Chicago, IL, USA Chicago, IL, USA
Priya R. Banerjee
Department of Physics
University at Buffalo, State University
Buffalo, NY, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)

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Preface
This volume on phase-separated biomolecular condensates comes at an opportune time. As

stated in the chapter by Mazarakos et al. (Chap. 1), “Phase-separated biomolecular con-
densates are revolutionizing our understanding of biology. Although phase separation is a
decidedly physical phenomenon, there are growing gaps between the biology and physics of
condensates.” The methods presented in this volume have contributed significantly to
narrowing these gaps, and it is thus timely to take stock of these contributions. Moreover,
as these methods have become established, we hope that this volume will help popularize
them and lead to broader applications. Lastly, many of the chapters take a forward look to
assess how these methods will evolve in the future. We thus hope that this volume will also
spur the development of new methods.
Table 1 gives a quick summary of the 22 chapters in this volume. The first five chapters
present theoretical and computational methods. The chapters by Mazarakos et al. (Chap. 1),
Najafi et al. (Chap. 2), and Lin et al. (Chap. 3) all describe methods for determining
binodals, that is, the equilibrium concentrations of the dilute and dense phases as a function
of temperature. Mazarakos et al. illustrate two traditional methods, Gibbs-ensemble Monte
Carlo and slab-geometry molecular dynamics simulations, as well a more recent method
called FMAP. In addition to binodals, this chapter also explains the calculation of the
interfacial tensions of condensates. It specifically shows how the finite sizes of simulation
systems affect binodals and interfacial tensions, and how these effects can be corrected.
Najafi et al. review field-theoretic simulations, which provide a powerful method for deter-
mining phase equilibria, initially for polymers and now adapted for intrinsically disordered
proteins (IDPs). Interestingly, field-theoretic simulations have been implemented in the
Gibbs ensemble, thereby reducing the computational cost for binodal determination. Lin
et al. also cover field-theoretic simulations, as well as an analytic method called random-
phase approximation and coarse-grained simulations. Illustrative results of the methods
include sequence effects on the binodal and on the multiphase organization of condensates.
Ginell and Holehouse (Chap. 4) review the stickers-and-spacers polymer model and show
how it can rationalize experimental observations on phase equilibria. Laghmach et al.
(Chap. 5) give an overview of modeling protein-RNA condensates at different scales,
from all-atom to ultra-coarse-grained. Modeling results include various morphologies of
condensates and nonequilibrium effects.
The next ten chapters present experimental methods for in vitro characterization of
biomolecular condensates. Quan et al. (Chap. 6) illustrate the application of fluorescence
lifetime imaging microscopy (FLIM) in characterizing the material states of condensates
formed by the TDP-43 low-complexity domain (TDP-43LCD). An interesting observation is
that trimethylamine N-oxide (TMAO), a chemical chaperone, enhances the phase separa-
tion of TDP-43LCD but inhibits its aggregation, by compacting the protein. Ganser et al.
(Chap. 7) describe single-molecule Förster resonance energy transfer (smFRET) and single-
molecule protein-induced fluorescence enhancement (smPIFE) experiments for probing
RNA conformational dynamics that can be regulated by interactions with proteins. These
methods potentially can be used to study RNA conformational dynamics inside condensates.
Two chapters, by Incicco et al. (Chap. 8) and by Alshareedah and Banerjee (Chap. 9),
describe the application of fluorescence correlation spectroscopy (FCS) in determining the
v
vi Preface
Table 1
Summary of chapters
Chapter Methods Applications Unique angle

Theory and computation
Mazarakos et al. Three simulation Binonals and Correction for finite system size
methods interfacial tension
Najafi et al. Field-theoretic Binodal of Sampling of polymer configurations;
simulations polyampholytes great potential for IDPs
Lin et al. Analytical theory; field- Binodals and Sequence dependence of IDPs in
theoretic and coarse- multiphase phase separation
grained simulations organization
Ginell and Stickers-and-spacers Rationalization of experimental observations on phase
Holehouse model equilibrium
Laghmach et al. Multi-scale modeling Protein-RNA Condensate morphology and
condensates nonequilibrium effects
Optical microscopy
Quan et al. FLIM TDP-43LCD TMAO enhances phase separation
condensates but inhibits aggregation, and can
thus tune the material state
Ganser et al. smFRET/PIFE Protein-RNA Potentially can probe interactions at
interactions single-molecule level inside
condensates
Incicco et al. FCS Concentrations; Theoretical background and
diffusion practical considerations
constants
Alshareedah and FCS Diffusion of single Calibration for accurately measuring
Banerjee molecules in diffusion constants
condensates
Zuo et al. DNA curtains Protein-DNA Real-time observation of DNA
condensates compaction
Pei et al. Condensate-aided Identification of High-throughput screening
enhancement of protein-protein
interactions interaction
partners
OT
Ghosh et al. Fluorescence microscopy Concentrations; OT is only method for measuring
and OT interfacial viscoelasticity of condensates
tension;
viscoelasticity
Other in-vitro methods
Li et al. Crystallization robot; Conditions for High-throughput screening
high-content analysis phase separation
(continued)
Preface vii
Table 1
(continued)
Chapter Methods Applications Unique angle

Wang and Fluorescence Condensates Condensates formed by folded
Hayer-Hartl microscopy; involved in proteins
transmission electron biogenesis of
microscopy carboxysome
Tollervey et al. Cryo-electron SPD-5; FUS; Identification of interaction
tomography G3BP1 networks
In-cell methods
Gruijs da Silva Sedimentation PTMs in RNP In vitro and in cell
and Dormann condensates
Reinkemeier Synthetic organelles for Reprogramming of Selective protein labeling in live cells
and Lemke GCE translation
Parmar and Single-molecule tracking Mobility of RNA Sub-micron biomolecular
weber polymerase condensates in live cells
Kim and Shin Optodroplets Plasmid preparation for delivering optodroplet
components into eukaryotic cells
Rademacher Optodroplets Image acquisition and analysis protocol for assessing HP1
et al. phase separation
Giesler et al. Mass balance imaging N-WASP/F-actin in Growth kinetics of condensates
C. elegans
Pandey et al. Super-resolution Intracellular single-molecule imaging methods for probing
microscopy; single- sub-diffraction condensates
molecule tracking
concentrations and diffusion constants of condensate components. The former focuses on

the theoretical background of FCS and practical considerations such as fluorophore selec-
tion; the latter focuses on diffusion constants and the necessary calibration experiments.
Combined, these two chapters provide the essential technical details, experimental proce-
dure, and data analysis schemes for the implementation of FCS for studying phase-separated
biomolecular condensates. The chapter by Zuo et al. (Chap. 10) reviews the application of a
method called DNA curtains in characterizing protein-DNA condensates. This method can
detect the conformational change of a single DNA molecule induced by phase separation.
Pei et al. (Chap. 11) illustrate condensate-aided enrichment of biomolecular interactions in
test tubes (CEBIT), whereby a protein of interest is fused to a scaffold protein that drives
condensate formation, and client proteins that are interaction partners of the protein of
interest are identified by their recruitment into the condensate. This method has the
potential for high-throughput screening of protein-protein interactions.
Ghosh et al. (Chap. 12) describe the application of fluorescence microscopy and optical
tweezers (OT) in measuring concentrations, interfacial tension, and viscoelasticity. Notably,
OT is the only method for measuring the viscoelasticity of phase-separated biomolecular
condensates. Li et al. (Chap. 13) introduce a high-throughput method for screening phase-
separation conditions. In this method, the authors use a crystallization robot to prepare a
viii Preface
large number of mixtures and then a high-content analysis system to rapidly select mixtures
that form condensates. Wang and Hayer-Hartl (Chap. 14) review their studies on conden-
sates involved in the biogenesis of carboxysomes, which are cytosolic bodies for photosyn-
thetic CO2 fixation. Interestingly, these condensates are formed by folded domains,
including Rubisco large and small subunits and multiple Rubisco small subunit-like domains
connected by flexible linkers. The methods used include transmission electron microscopy
in both negative-staining and cryo conditions. The chapter by Tollervey et al. (Chap. 15)
describes the application of cryo-electron tomography in a variety of reconstituted biomo-
lecular condensates. This method is capable of revealing interaction networks inside
condensates.
The last seven chapters present methods that enable in-cell characterization of biomo-
lecular condensates. Gruijs da Silva and Dormann (Chap. 16) outline sedimentation assays
in vitro and in cell to analyze how the phase behaviors of ribonucleoproteins (RNPs) can be
modulated by post-translational modifications (PTMs) such as phosphorylation. Given that
many signaling proteins harbor multiple sites for PTMs, these assays provide simple yet
useful tools to study such processes. Reinkemeier and Lemke (Chap. 17) describe an elegant
method to generate synthetic organelles in live cells that perform genetic code expansion
(GCE), protein-selective non-canonical amino-acid incorporation, and subsequent labeling
by small-molecule fluorophores. Parmar and Weber (Chap. 18) review single-molecule
tracking for quantifying the mobility of a bacterial RNA polymerase in transcriptional
condensates. This technique is particularly useful for studying sub-micron biomolecular
condensates in live cells. Kim and Shin (Chap. 19) describe the optodroplet assay and show
efficient ways to generate and deliver plasmids into eukaryotic cells. Rademacher et al.
(Chap. 20) demonstrate how to use this assay to assess the phase-separation propensity of
the heterochromatin protein HP1 in the nucleus of live cells. Together, these two chapters
provide a detailed recipe for using optogenetic approaches to study condensates in live cells,
from plasmid preparation to image acquisition and analysis. Giesler et al. (Chap. 21) review
mass balance imaging (MBI) for determining the phase behaviors and dissecting the kinetic
properties of multi-component condensates. Finally, Pandey et al. (Chap. 22) discuss
intracellular single-molecule imaging methods (super-resolution microscopy and single-
molecule tracking). Carefully interpreted, these methods yield quantitative biophysical
parameters at length scales inaccessible by conventional assays.
The 22 chapters collectively provide a broad repertoire of theoretical, computational,
and experimental methods to quantitatively interrogate the properties of phase-separated
biomolecular condensates in diverse systems. As new discoveries of condensates are being
made continuously in this burgeoning field, these chapters by no means provide a complete
list of methods that are applicable in condensate studies. Rather they represent a collection
of well-established tools that can be readily applied to existing as well as newly discovered
condensate systems. We expect that this collection will catalyze quantitative studies of both
biophysical properties and biological functions of complex biomolecular condensates.
We thank all the authors for their valuable contributions to this volume on phase-
separated biomolecular condensates.
Chicago, IL, USA Huan-Xiang Zhou

Buffalo, NY, USA Priya R. Banerjee
Chicago, IL, USA Jan-Hendrik Spille
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Calculating Binodals and Interfacial Tension of Phase-Separated
Condensates from Molecular Simulations with Finite-Size Corrections . . . . . . . . 1
Konstantinos Mazarakos, Sanbo Qin, and Huan-Xiang Zhou
2 Field-Theoretic Simulation Method to Study the Liquid–Liquid
Phase Separation of Polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Saeed Najafi, James McCarty, Kris T. Delaney,
Glenn H. Fredrickson, and Joan-Emma Shea
3 Numerical Techniques for Applications of Analytical Theories
to Sequence-Dependent Phase Separations of Intrinsically
Disordered Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Yi-Hsuan Lin, Jonas Wessén, Tanmoy Pal, Suman Das,
and Hue Sun Chan
4 An Introduction to the Stickers-and-Spacers Framework as Applied
to Biomolecular Condensates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Garrett M. Ginell and Alex S. Holehouse
5 Multiscale Modeling of Protein-RNA Condensation
in and Out of Equilibrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Rabia Laghmach, Isha Malhotra, and Davit A. Potoyan
6 Fluorescence Lifetime Imaging Microscopy of Biomolecular
Condensates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
My Diem Quan, Shih-Chu Jeff Liao, Josephine C. Ferreon,
and Allan Chris M. Ferreon
7 Single-Molecule Fluorescence Methods to Study Protein-RNA
Interactions Underlying Biomolecular Condensates . . . . . . . . . . . . . . . . . . . . . . . . . 149
Laura R. Ganser, Yingda Ge, and Sua Myong
8 Fluorescence Correlation Spectroscopy and Phase Separation. . . . . . . . . . . . . . . . . 161
Juan Jeremı́as Incicco, Debjit Roy, Melissa D. Stuchell-Brereton,
and Andrea Soranno
9 Measurement of Protein and Nucleic Acid Diffusion Coefficients Within
Biomolecular Condensates Using In-Droplet Fluorescence
Correlation Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Ibraheem Alshareedah and Priya R. Banerjee
10 Single-Molecule Imaging of the Phase Separation-Modulated
DNA Compaction to Study Transcriptional Repression. . . . . . . . . . . . . . . . . . . . . . 215
Linyu Zuo, Luhua Lai, and Zhi Qi
11 Phase Separation-Based Biochemical Assays for Biomolecular
Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Gaofeng Pei, Min Zhou, Weifan Xu, Jing Wang, and Pilong Li
ix
x Contents
12 Determining Thermodynamic and Material Properties of Biomolecular

Condensates by Confocal Microscopy and Optical Tweezers . . . . . . . . . . . . . . . . . 237
Archishman Ghosh, Divya Kota, and Huan-Xiang Zhou
13 A High-Throughput Method to Profile Protein Liquid-Liquid
Phase Separation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Yichen Li, Jinge Gu, Cong Liu, and Dan Li
14 Phase Separation of Rubisco by the Folded SSUL Domains
of CcmM in Beta-Carboxysome Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Huping Wang and Manajit Hayer-Hartl
15 Cryo-Electron Tomography of Reconstituted Biomolecular
Condensates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Fergus Tollervey, Xiaojie Zhang, Mainak Bose, Jenny Sachweh,
Jeffrey B. Woodruff, Titus M. Franzmann, and Julia Mahamid
16 Sedimentation Assays to Assess the Impact of Posttranslational
Modifications on Phase Separation of RNA-Binding Proteins
In Vitro and In Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Lara A. Gruijs da Silva and Dorothee Dormann
17 Synthetic Organelles for Multiple mRNA Selective Genetic Code
Expansions in Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Christopher D. Reinkemeier and Edward A. Lemke
18 Single-Molecule Tracking of RNA Polymerase In and Out
of Condensates in Live Bacterial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Baljyot Singh Parmar and Stephanie C. Weber
19 An Optogenetic Toolkit for the Control of Phase Separation
in Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Chaelim Kim and Yongdae Shin
20 Assessing the Phase Separation Propensity of Proteins in Living
Cells Through Optodroplet Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Anne Rademacher, Fabian Erdel, Robin Weinmann,
and Karsten Rippe
21 Mass Balance Imaging: A Phase Portrait Analysis for Characterizing
Growth Kinetics of Biomolecular Condensates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
Jan Geisler, Victoria Tianjing Yan, Stephan Grill,
and Arjun Narayanan
22 Characterizing Properties of Biomolecular Condensates Below
the Diffraction Limit In Vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Ganesh Pandey, Alisha Budhathoki, and Jan-Hendrik Spille
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
Contributors
IBRAHEEM ALSHAREEDAH • Department of Physics, University at Buffalo SUNY, Buffalo, NY,

USA
PRIYA R. BANERJEE • Department of Physics, University at Buffalo SUNY, Buffalo, NY, USA
MAINAK BOSE • Developmental Biology Unit, European Molecular Biology Laboratory
(EMBL), Heidelberg, Germany
ALISHA BUDHATHOKI • Department of Physics, University of Illinois at Chicago, Chicago, IL,
USA
HUE SUN CHAN • Department of Biochemistry, University of Toronto, Toronto, ON, Canada
SUMAN DAS • Department of Biochemistry, University of Toronto, Toronto, ON, Canada
KRIS T. DELANEY • Materials Research Laboratory, University of California, Santa Barbara,
CA, USA
DOROTHEE DORMANN • Johannes Gutenberg-Universit€ at (JGU), Biocenter, Institute of
Molecular Physiology, Mainz, Germany; Institute of Molecular Biology (IMB), Mainz,
Germany
FABIAN ERDEL • MCD, Centre de Biologie Intégrative (CBI), University of Toulouse, CNRS,
Toulouse, France
ALLAN CHRIS M. FERREON • Department of Pharmacology and Chemical Biology, Baylor
College of Medicine, Houston, TX, USA
JOSEPHINE C. FERREON • Department of Pharmacology and Chemical Biology, Baylor College
of Medicine, Houston, TX, USA
TITUS M. FRANZMANN • Center for Molecular and Cellular Bioengineering, Biotechnology
Center, Technische Universit€at Dresden, Dresden, Germany
GLENN H. FREDRICKSON • Materials Research Laboratory, University of California, Santa
Barbara, CA, USA; Department of Chemical Engineering, University of California,
Santa Barbara, CA, USA
LAURA R. GANSER • Department of Biophysics, Johns Hopkins University, Baltimore, MD,
USA
YINGDA GE • Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA
JAN GEISLER • Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany
ARCHISHMAN GHOSH • Department of Chemistry, University of Illinois at Chicago, Chicago,
IL, USA
GARRETT M. GINELL • Department of Biochemistry and Molecular Biophysics, Washington
University School of Medicine, St. Louis, MO, USA; Center for Science & Engineering of
Living Systems (CSELS), Washington University in St. Louis, St. Louis, MO, USA
STEPHAN GRILL • Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany; Center for Systems Biology Dresden, Dresden, Germany; Cluster of Excellence
Physics of Life, TU Dresden, Dresden, Germany
LARA A. GRUIJS DA SILVA • Johannes Gutenberg-Universit€ a t (JGU), Biocenter, Institute of
Molecular Physiology, Mainz, Germany; Graduate School of Systemic Neurosciences (GSN),
Planegg-Martinsried, Germany
xi
xii Contributors
JINGE GU • Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute

of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China; University of the
Chinese Academy of Sciences, Beijing, China
MANAJIT HAYER-HARTL • Membrane protein biosynthesis and quality control, MRC
Laboratory of Molecular Biology, Cambridge, United KingdomFrancis Crick Avenue,
ALEX S. HOLEHOUSE • Department of Biochemistry and Molecular Biophysics, Washington
University School of Medicine, St. Louis, MO, USA; Center for Science & Engineering of
Living Systems (CSELS), Washington University in St. Louis, St. Louis, MO, USA
JUAN JEREMÍAS INCICCO • Department of Biochemistry and Molecular Biophysics, Washington
University in St Louis, St. Louis, MO, USA; Center for Science and Engineering of Living
Systems (CSELS), Washington University in St Louis, St. Louis, MO, USA
CHAELIM KIM • Interdisciplinary Program in Bioengineering, Seoul National University,
Seoul, Republic of Korea
DIVYA KOTA • Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA
RABIA LAGHMACH • Department of Chemistry, Iowa State University, Ames, IA, USA
LUHUA LAI • Center for Quantitative Biology and Peking-Tsinghua Center for Life Sciences,
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China;
BNLMS, College of Chemistry and Molecular Engineering, Peking University, Beijing,
China
EDWARD A. LEMKE • Biocentre, Departments of Biology and Chemistry, Johannes Gutenberg
University Mainz, Mainz, Germany; Institute of Molecular Biology gGmbH, Mainz,
Germany
DAN LI • Bio-X Institutes, Key Laboratory for the Genetics of Developmental and
Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University,
Shanghai, China; Bio-X-Renji Hospital Research Center, Renji Hospital, School of
Medicine, Shanghai Jiao Tong University, Shanghai, China; Zhangjiang Institute for
Advanced Study, Shanghai Jiao Tong University, Shanghai, China
PILONG LI • Beijing Advanced Innovation Center for Structural Biology & Frontier
Research Center for Biological Structure, School of Life Sciences, Tsinghua University,
Beijing, China; Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing,
China
YICHEN LI • Bio-X Institutes, Key Laboratory for the Genetics of Developmental and
Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University,
Shanghai, China
SHIH-CHU JEFF LIAO • ISS, Inc., Champaign, IL, USA
YI-HSUAN LIN • Department of Biochemistry, University of Toronto, Toronto, ON, Canada;
Molecular Medicine, Hospital for Sick Children, Toronto, ON, Canada
CONG LIU • Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute
of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China; University of the
Chinese Academy of Sciences, Beijing, China
JULIA MAHAMID • Structural and Computational Biology Unit, European Molecular Biology
Laboratory (EMBL), Heidelberg, Germany
ISHA MALHOTRA • Department of Chemistry, Iowa State University, Ames, IA, USA
KONSTANTINOS MAZARAKOS • Department of Physics, University of Illinois at Chicago,
Chicago, IL, USA
JAMES MCCARTY • Department of Chemistry, Western Washington University, Bellingham,
WA, USA
Contributors xiii
SUA MYONG • Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA
SAEED NAJAFI • Department of Chemistry and Biochemistry, University of California, Santa
Barbara, CA, USA; Materials Research Laboratory, University of California, Santa
Barbara, CA, USA
ARJUN NARAYANAN • Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany; Center for Systems Biology Dresden, Dresden, Germany; Max Planck Institute for
the Physics of Complex Systems, Dresden, Germany
TANMOY PAL • Department of Biochemistry, University of Toronto, Toronto, ON, Canada
GANESH PANDEY • Department of Physics, University of Illinois at Chicago, Chicago, IL, USA
BALJYOT SINGH PARMAR • Department of Physics, McGill University, QC, Canada
GAOFENG PEI • Beijing Advanced Innovation Center for Structural Biology & Frontier
China
DAVIT A. POTOYAN • Department of Chemistry, Iowa State University, Ames, IA, USA
ZHI QI • Center for Quantitative Biology and Peking-Tsinghua Center for Life Sciences,
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
SANBO QIN • Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA
MY DIEM QUAN • Department of Pharmacology and Chemical Biology, Baylor College of
Medicine, Houston, TX, USA
ANNE RADEMACHER • Division of Chromatin Networks, German Cancer Research Center
(DKFZ) and Bioquant, Heidelberg, Germany
CHRISTOPHER D. REINKEMEIER • Biocentre, Departments of Biology and Chemistry, Johannes
Gutenberg University Mainz, Mainz, Germany; Institute of Molecular Biology gGmbH,
Mainz, Germany
KARSTEN RIPPE • Division of Chromatin Networks, German Cancer Research Center
DEBJIT ROY • Department of Biochemistry and Molecular Biophysics, Washington University
in St Louis, St. Louis, MO, USA; Center for Science and Engineering of Living Systems
(CSELS), Washington University in St Louis, St. Louis, MO, USA
JENNY SACHWEH • Structural and Computational Biology Unit, European Molecular Biology
Laboratory (EMBL), Heidelberg, Germany; Max Planck Institute of Biophysics, Frankfurt
am Main, Germany
JOAN-EMMA SHEA • Department of Chemistry and Biochemistry, University of California,
Santa Barbara, CA, USA; Department of Physics, University of California Santa
Barbara, Santa Barbara, CA, USA
YONGDAE SHIN • Interdisciplinary Program in Bioengineering, Seoul National University,
Seoul, Republic of Korea; Department of Mechanical Engineering, Seoul National
University, Seoul, Republic of Korea; Institute of Advanced Machines and Design, Seoul
National University, Seoul, Republic of Korea
ANDREA SORANNO • Department of Biochemistry and Molecular Biophysics, Washington
University in St Louis, St. Louis, MO, USA; Center for Science and Engineering of Living
Systems (CSELS), Washington University in St Louis, St. Louis, MO, USA
JAN-HENDRIK SPILLE • Department of Physics, University of Illinois at Chicago, Chicago, IL,
USA
xiv Contributors
MELISSA D. STUCHELL-BRERETON • Department of Biochemistry and Molecular Biophysics,

Washington University in St Louis, St. Louis, MO, USA; Center for Science and
Engineering of Living Systems (CSELS), Washington University in St Louis, St. Louis, MO,
USA
FERGUS TOLLERVEY • Structural and Computational Biology Unit, European Molecular
Biology Laboratory (EMBL), Heidelberg, Germany; Collaboration for Joint PhD Between
EMBL and Heidelberg University Faculty of Biosciences, Heidelberg, Germany
HUPING WANG • Department of Cellular Biochemistry, Max Planck Institute of
Biochemistry, Martinsried, GermanyAm Klopferspitz 18,
JING WANG • Beijing Advanced Innovation Center for Structural Biology & Frontier
China
STEPHANIE C. WEBER • Department of Physics, McGill University, QC, Canada; Department
of Biology, McGill University, QC, Canada
ROBIN WEINMANN • Division of Chromatin Networks, German Cancer Research Center
JONAS WESSÉN • Department of Biochemistry, University of Toronto, Toronto, ON, Canada
JEFFREY B. WOODRUFF • Department of Cell Biology, Department of Biophysics, UT
Southwestern Medical Center, Dallas, TX, USA
WEIFAN XU • Beijing Advanced Innovation Center for Structural Biology & Frontier
China
VICTORIA TIANJING YAN • Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany; BIOTEC, TU Dresden, Dresden, Germany
XIAOJIE ZHANG • Structural and Computational Biology Unit, European Molecular Biology
Laboratory (EMBL), Heidelberg, Germany
HUAN-XIANG ZHOU • Department of Chemistry, University of Illinois at Chicago, Chicago,
IL, USA; Department of Physics, University of Illinois at Chicago, Chicago, IL, USA
MIN ZHOU • Beijing Advanced Innovation Center for Structural Biology & Frontier
China
LINYU ZUO • Center for Quantitative Biology and Peking-Tsinghua Center for Life Sciences,
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
Chapter 1
Calculating Binodals and Interfacial Tension

of Phase-Separated Condensates from Molecular
Simulations with Finite-Size Corrections
Konstantinos Mazarakos, Sanbo Qin, and Huan-Xiang Zhou
Abstract
We illustrate three methods for calculating the binodals of phase-separated condensates from molecular
simulations. Because molecular simulations can only be carried out for small system sizes, correction for
finite sizes may be required for making direct comparison between calculated results and experimental data.
We first summarize the three methods and then present detailed implementation of each method on a
Lennard-Jones fluid. In the first method, chemical potentials are calculated over a range of particle densities
in canonical-ensemble simulations; the densities of the dilute and dense phases at the given temperature are
then found by a Maxwell equal-area construction. In Gibbs-ensemble Monte Carlo, the exchange between
separated dilute and dense phases is simulated to obtain their densities. Lastly, slab-geometry molecular
dynamics simulations model the dilute and dense phases in coexistence and yield not only their densities but
also their interfacial tension. The three types of simulations are carried out for a range of system sizes, and
the results are scaled to generate the binodals corrected for finite system sizes. Size-corrected interfacial
tension is also produced from slab-geometry molecular dynamics simulations.
Key words Phase separation, Biomolecular condensates, Binodals, Interfacial tension, Lennard-Jones
fluid, FMAP, Monte-Carlo simulation, Molecular dynamics simulation, Finite-size scaling
1 Introduction
Phase-separated biomolecular condensates are revolutionizing our

understanding of biology. Although phase separation is a decidedly
physical phenomenon, there are growing gaps between the biology
and physics of condensates. Computational studies have filled some
of these gaps. For example, using Gibbs-ensemble Monte Carlo
simulations of patchy particles, we have shown that disparate effects
of macromolecular regulators observed on the phase equilibrium of
a variety of biomolecular condensates can be recapitulated by tun-
ing the strength of interactions between the regulators and the
condensate driver proteins [1]. This prediction motivated
Huan-Xiang Zhou et al. (eds.), Phase-Separated Biomolecular Condensates: Methods and Protocols,
Methods in Molecular Biology, vol. 2563, https://doi.org/10.1007/978-1-0716-2663-4_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023
1
2 Konstantinos Mazarakos et al.
experimental verification and led to the classification of all macro-

molecular regulators into three architypes [2]. Recently, using slab-
geometry molecular dynamics simulations of particle and chain
models, we have predicted that macromolecular regulators have
matching effects on the critical temperature for phase separation
and the interfacial tension between the phases [3]. Coarse-grained
simulations have also yielded insight into the sequence dependence
of the phase equilibrium of intrinsically disordered proteins [4–7].
In their simplest form, biomolecular condensates are a homo-
geneous, dense phase in coexistence with a surrounding dilute
phase. In many respects, this coexistence between the dilute and
dense phases is similar to that between the vapor and liquid phases
of a simple molecular fluid like water [8]. In both cases, the system
prefers the two separate phases, instead of a single phase, because
such a single phase would have a higher free energy. The coexis-
tence condition is satisfied when the temperature (T), pressure ( p),
and chemical potential (μ) of each species are equal between the
two phases. This condition allows for the determination of the
disparate densities of each species in the two phases. The depen-
dence of these densities on the temperature is called a binodal. As
the temperature is increased, the two phases become more and
more similar to each other, and correspondingly, the two branches
of the binodal approach each other and, finally, merge at the critical
temperature (Tc). Above the critical temperature, the system no
longer phase-separates. At the interface between two coexisting
phases, the disparity in density and hence in intermolecular inter-
actions generates an interfacial tension (γ).
Binodals can be calculated by designing different routes to
satisfy the equality in T, p, and μ between the two phases. In this
chapter, we illustrate three such routes. In the first route, chemical
potentials are calculated over a range of densities in canonical-
ensemble simulations [9]. The densities of the dilute and dense
phases at the given temperature are then found by a Maxwell equal-
area construction, which guarantees equality in chemical potential
and pressure. In Gibbs-ensemble Monte Carlo, the exchange
between separated dilute and dense phases is simulated to obtain
their densities [10]. The exchange in volume guarantees equality in
pressure, whereas the swap in particles guarantees equality in chem-
ical potential. Lastly, slab-geometry molecular dynamics simula-
tions model the dilute and dense phases in coexistence, directly
mimicking physical systems and naturally satisfying the equality in
T, p, and μ [11]. In addition to the densities of the two phases, these
simulations also yield the interfacial tension.
Maxwell [12] recognized that, for a real fluid with intermolec-
ular interactions, the pressure has a nonmonotonic dependence on
the volume (V) at low temperature (i.e., < Tc). The nonmonotonic
portion is called a van der Waals loop. The vapor and liquid den-
sities can be identified by a horizontal line that bisects the van der
Calculating Binodals and Interfacial Tension of Phase-Separated. . . 3
Waals loop with equal areas above and below. The same idea has
been extended to the common-tangent construction when the
dependence of the Helmholtz free energy on the density is known
[8] and to an equal-area construction when the dependence of the
chemical potential on the density is known [9]. These constructions
have been used to calculate binodals for proteins [9, 13–15]. The
hard problem is the calculation of the free energy or chemical
potential. We have developed a powerful method called FMAP, or
fast Fourier transform-based modeling of atomistic protein-protein
interactions, for calculating the chemical potentials of protein solu-
tions and used FMAP to determine the binodal for γ-crystallin
[9]. FMAP has been extended to calculate the second virial coeffi-
cients (B2) [16] and cross second virial coefficients (B23) [17] for
proteins represented at the all-atom level in implicit solvent. Both
of these coefficients give qualitative indications on the phase equi-
librium. The second virial coefficient is related to the critical tem-
perature for the phase separation of the driver species: a more
negative B2 corresponds to a higher Tc. The relative values of B2
and B23 measure the relative strength of self-interaction and cross-
species interaction. The latter quantity is a key determinant for the
classification of macromolecular regulators into three architypes
[2], and may also be important factor for the multiphase organiza-
tion of multicomponent biomolecular condensates [3]. Recently,
field-theoretic simulations have been used to calculate the chemical
potentials of intrinsically disordered proteins [7].
Gibbs-ensemble Monte Carlo has been used to study the phase
equilibrium of biomolecular condensates and complex coacervates,
with the macromolecular components modeled as spherical parti-
cles [1, 2, 18] or chains [19, 20]. As just noted, our own such
studies have led to the identification of the relative strength of
intermolecular interactions as a key determinant for the classifica-
tion of macromolecular regulators into three architypes [1, 2]. For
chains, the swap between two boxes, specifically the insertion of a
chain into a dense box, is difficult to achieve. This difficulty has led
to the elimination of swaps for chains, allowing such moves only for
ions [19, 20]. Recently, field-theoretic simulations have been used
to implement the Gibbs ensemble, allowing thorough sampling of
chain configurations and swaps between the boxes [21].
Slab-geometry molecular dynamics simulations have been
applied to systems modeled at different levels of details, from
point particles to proteins in explicit solvent [3, 4, 22–25]. Our
own such study has resulted in the prediction that macromolecular
regulators have matching effects on the critical temperature and the
interfacial tension [3]. Another interesting prediction from the slab
method is a correlation between the theta temperature of a single
polymer chain and the critical temperature for phase separation
[23]. Although slab-geometry molecular dynamics simulations
have been performed on proteins in explicit solvent, they have
failed to reach equilibration between the phases [24].
All molecular simulations can only be done on a system size far

smaller than the experimental counterpart, as the computational
time grows significantly with system size. Therefore, a real concern
is the error caused by finite system size. While finite-size effects are a
problem for simulations in general, they are especially so for calcu-
lating properties near the critical point. Because the correlation
length grows to infinity at the critical point, when near the critical
point, the finite size of a simulation system can easily fall far short of
the correlation length, leading to serious errors in calculated prop-
erties [26]. In cases where Maxwell construction is used, the system
size also affects the shape of the van der Waals loop: with increasing
system size, the loop becomes less prominent or even flat in the
middle [27, 28], making it difficult to precisely locate the densities
of the dilute and dense phases. So for this practical reason alone,
simulation studies that rely on Maxwell construction have to limit
the system size. Fortunately, finite-size scaling, based on simula-
tions over a range of system sizes, allows the results to be extra-
polated to infinite size. While finite-size scaling has been widely
used in the studies of simple particle systems [29], finite-size effects
have received little attention in simulation studies of bimolecular
condensates. In this chapter, we illustrate how scaling can be done
in each of the three methods. We hope to both call attention to the
finite-size issue and provide some guidance on its magnitude and
correction. Slab-geometry molecular dynamics simulations are
found to have the strongest dependence on system size, likely
related to the fact that interfaces are present in these simulation
systems.
2 Summary of Methods
We illustrate the three methods for calculating binodals on a

Lennard-Jones fluid. This fluid comprises particles that interact
via the potential
6
r 12 r
uLJ ðr Þ ¼ 4ε ð1Þ
σ σ
where r denotes the interparticle distance. The parameter ε repre-
sents the depth of the energy well, whereas σ is the contact distance,
that is, the r value at which the potential function crosses the r axis
(Fig. 1). To reduce computational cost, a cutoff, rcut, is introduced,
such that the potential is modified to

uLJ ðr Þ uLJ ðr cut Þ, r < r cut
uðr Þ ¼ ð2Þ
0, r r cut
Fig. 1 The Lennard-Jones (LJ) potential and its shifted version, for the interaction
between a pair of particles. The inset shows a zoom into a small range of the
interparticle distance around the cutoff rcut ¼ 3, showing that LJ shifted is zero
at r ¼ rcut. For r > rcut, LJ shifted is fixed at 0. Note that, at r ¼ rcut, although LJ
shifted is continuous, its slope and hence the interparticle force have a
discontinuity
Note that, when the distance is within the cutoff, the potential
is shifted up to ensure continuity at r ¼ rcut (Fig. 1). For simplicity,
we measure length, energy, interfacial tension, and temperature in
units of σ, ε, ε/σ 2, and ε/kB, respectively, where kB is the Boltz-
mann constant. p Inffiffiffiffiffiffiffiffiffiffiffiffiffi
molecular
ffi dynamics simulations, we measure
time in units of mσ 2 =ε, where m is the mass of the particles. We
chose rcut ¼ 3 for all the results reported here.
2.1 Chemical In the canonical ensemble, the chemical potential can be decom-
Potential by Brute- posed into
Force Insertion μ ¼ μid þ μex ð3Þ
The ideal part,
μid ¼ kB T ln ðρ=ρ0 Þ ð4Þ
is that of an ideal gas, where ρ denotes the particle number density,
and ρ0 is an unimportant constant (see Note 1). The excess part,
μex, arises from interparticle interactions. According to Widom
[30], μex can be expressed as

e βμex ¼ e βU I ð5Þ
where β ¼ 1/kBT;
X
N
UI ¼ uIi ð6Þ
i¼1
Fig. 2 Illustration of calculating the chemical potential by Widom insertion. (a)

The inserted particle has interaction energies uI1, uI2, . . ., and uIN with particles
1, 2, . . ., and N in the system under constant N, V, and T. Note that the insertion
is fictitious because the inserted particle merely probes its interactions with the
N particles but does not affect their motions. (b) Monte Carlo simulations of the
N-particle system. A particle is randomly selected from the list of N, and a
displacement is then proposed, with uniform probability within a cube centered
at the original position and with side length 2lmax (dashed box). The would-be
change in the total energy, ΔU, of the system is calculated, and the proposed
displacement is accepted with the probability shown
is the sum of the interaction energies between an inserted particle

and all the N particles of the system under constant N, volume (V),
and T; and h. . .i denotes a canonical-ensemble average (Fig. 2a).
The direct way to implement Eq. (5) is by brute-force inser-
tion, that is, randomly inserting into the volume V over and over.
To carry out the average h. . .i, the configurations of the N-particle
system need to be sampled from simulations at constant N, V, and
T. Note that the particle insertion is fictitious as the inserted parti-
cle does not affect the configurations of the N particles. Here we
chose Monte Carlo simulations for configurational sampling at
constant N, V, and T (Fig. 2b). At each step, a particle is randomly
picked from the list of N for proposing a displacement. The dis-
placement is randomly selected inside a cube centered at the origi-
nal position and with side length 2lmax. The would-be change in the
total energy, ΔU, of the system is calculated, and the proposed
displacement is accepted if the Boltzmann factor eβΔU is greater
than a random number uniformly distributed between 0 and 1. If
not accepted, the selected particle repeats its old position.
2.2 Chemical While brute-force insertion is widely used for simple fluids such as
Potential by FMAP that comprising Lennard-Jones particles, it is too expensive to use
for protein solutions when the proteins are modeled with atomic
details. For the latter we developed the FMAP method [31]. The
key idea of FMAP is to express intermolecular interactions as cross-
correlation functions, and then use fast Fourier transform (FFT) to
speed up the evaluation of these functions.
Terms of the interaction potential usually have the form
u12 ðrÞ ¼ q 1 q 2 ϕðrÞ ð7Þ

For example, for Coulomb interactions, q1 and q2 represent the
charges of the particles and ϕ(r) ¼ 1/r. The Lennard-Jones poten-
tial can likewise be cast into this form, where q1 ¼ q2 ¼ 1. In the
following, we use the language for Coulomb interactions but
emphasize that the results apply to other types of interactions. We
can interpret the interaction of Eq. (7) as a product of the electric
potential f(r) ¼ q1ϕ(r) of particle 1 with the charge q2 of particle
2. For a particle inserted at position xI, the total interaction energy
with an N-particle system (see Eq [6]) is
X
N
U I ð xI Þ ¼ f ðxi xI Þq i
i¼1
Z X
N
¼ d 3 x0 f ðx0 xI Þ q i δðx0 xi Þ
i¼1
Z X
N
¼ d 3 xf ðxÞ q i δðx þ xI xi Þ
i¼1
Z
¼ d 3 xf ðxÞg ðx þ xI Þ ð8Þ
where δ(x) is a three-dimensional delta function and g(x) is the

charge density of the N-particle system:
X
N
g ðxÞ ¼ q i δðx xi Þ ð9Þ
i¼1
We can recognize the right-hand side of Eq. (8) as the cross-

correlation function of the electric potential f(x) of the inserted
particle and the charge density g(x) of the N-particle system. While
in direct space the cross-correlation function involves a three-
dimensional integral, its Fourier transform is a simple product:
F fU I ðxI Þg ¼ F ðkÞG ðkÞ ð10Þ
where F(k) and G(k) are Fourier transforms of f(x) and g(x),
respectively, and * denotes complex conjugate.
The FMAP procedure is illustrated in Fig. 3. First, the potential
function f(x) and the charge density g(x) are mapped to a cubic
grid. Mapping for f(x) simply means evaluating this function at
each grid point; mapping for g(x) involves distributing the point
charge to the vertices of the enclosing voxel. The Fourier trans-
forms of f(x) and g(x) as well as the inverse Fourier transform of
F*(k)G(k) are then calculated by FFT. The latter inverse Fourier
transform is the energy function UI(xI), which contains the values
of the interaction energy at all the grid points. FMAP is thus
Fig. 3 Illustration of FMAP for a two-dimensional system. (a) Top: the “potential” function f(x) of the inserted
particle, shown in a color scale with red and blue representing positive and negative values, respectively.
Bottom: the “charge” density g(x) of the N-particle system. The black dot displays the position of a particle,
and the light red shading represents the smear of a point charge. (b) Top: the discretized version of panel (a)
top, on a 16 16 grid, with a grid spacing of 0.5. Bottom: the discretized version of panel (b) bottom. The
point charge of each particle is distributed to the four nearest pixels. (c) Top: after the Fourier transform (FT) of
f(x) and g(x), the product F*(k)G(k) undergoes inverse Fourier transform (IFT) to yield the energy function UI(xI),
for the interaction of the inserted particle at position xI with the N-particle system. Bottom: with an increase in
grid points from 16 16 to 80 80, the energy function becomes very accurate
equivalent to brute-force insertion at all the grid points, but at a

much lower computational cost.
The discretization for FFT incurs numerical errors, but we can
compensate for these errors by adjusting the potential function. For
example, for the Lennard-Jones potential, at a grid space of 0.1,
discretization errors are minimized by increasing ε by a factor of
1.06 and decreasing σ by a factor of 6/6.06 [9]. Compensatory
factors have likewise been obtained for potential functions model-
ing atomistic proteins [31].
2.3 Maxwell Once the chemical potential at a given temperature is obtained as a

Construction for function of particle density, we can use a Maxwell construction to
Determining Binodals determine the densities of the dilute and dense phases [9]. Max-
well’s original construction was done for the pressure as a function
of volume [12], where equality in the areas above and below a line
bisecting the van der Waals loop ensures equality in chemical
potential between the two phases. We found that a corresponding
construction works for the chemical potential as a function of
particle density [9]. Here equality in areas above and below a line
bisecting the van der Waals loop ensures equality in pressure
between the two phases.
Fig. 4 The excess chemical potential, calculated by FMAP, over a range of

particle densities. (a) Fifteen systems, with increasing number of particles in the
same volume, are each simulated at constant N, V, T. (b) The excess chemical
potential at 15 densities, and its fit to a 5th-order polynomial. The number of
particles is Ni ¼ 30i, where i ¼ 1–15; the side length of each cubic box is L ¼ 8.
The FMAP calculations are done on an 80 80 80 grid, with grid spacing at
0.1. To compensate for discretization errors, the Lennard-Jones potential is
adjusted, with ε increased by a factor of 1.06 and σ decreased by a factor of
6/6.06 [9]. (c) Maxwell construction based on the full chemical potential as a
function of the particle density. The equal-area chemical potential, indicated by a
dotted horizontal line, is calculated using the van der Waals loop in which the
excess chemical potential is given by the polynomial fit. The left-most and right-
most intersections of the line with the loop are the densities of the dilute and
dense phases (indicated by circles)
In practice, using the method described in Subheading 2.1 or

2.2, we determine the excess chemical potentials for a series of
systems with increasing number of particles in the same volume
(Fig. 4a). We then fit the dependence of the excess chemical poten-
tial on particle density to a 5th-order polynomial (Fig. 4b; see Note
2). Finally, the polynomial fit is added to the ideal part to yield the
full chemical potential, and a Maxwell construction is carried out to
find the chemical potential and the densities of the two phases at
coexistence (Fig. 4c). Specifically, a horizontal line that bisects the
van der Waals loop with equal areas above and below is determined.
The intercept of the line with the chemical potential axis is the
chemical potential at coexistence, and the densities at the left-most
and right-most intersections of the line with the loop are those of
the dilute and dense phases, respectively.
Two other notable positions on the van der Waals loop are the
maximum and minimum (Fig. 4c). The densities at these two
extrema define the so-called spinodal. Between these two densities,

the chemical potential is a decreasing function of density, which is
counter to the usual trend for physical systems. Systems with den-
sities inside the spinodal are thus unstable.
Another often used construction, known as common tangent,
applies to the Helmholtz free energy per unit volume, f F/V, as a
function of ρ. We have shown that the common-tangent construc-
tion is closely related to our equal-area construction applied to μ as
a function of ρ [8]. In essence, μ ¼ ∂f∂ρ T
, so a common tangent in
the ρ f plane corresponds to a horizontal line in the ρ μ plane,
and both lead to the equality in chemical potential between the two
phases. A common intercept in the ρ f plane corresponds to equal
areas in the ρ μ plane, and both lead to the equality in pressure
between the two phases.
2.4 Gibbs-Ensemble Gibbs-ensemble Monte Carlo was designed to obtain the binodals
Monte Carlo of particle systems away from the critical point [10]. This method
entails the simulations of two separated but coupled systems
(Fig. 5a), which are initially identical, with a density in the unstable
region, but after equilibration end up with the two densities of the
dilute and dense phases, respectively (Fig. 5b). The temperature,
total particle number (N), and total volume of the two systems are
held constant. The simulations consist of three types of Monte
Carlo moves (Fig. 5a). Particle displacement equilibrates particles
in each system (Fig. 5a top). Volume exchange results in equality in
pressure between the systems (Fig. 5a middle), whereas particle
swap results in equality in chemical potential (Fig. 5a bottom).
The simulations are advanced in Monte Carlo cycles, each of
which is made up of a fixed number of moves distributed among
particle displacements, volume exchanges, and particle swaps. In a
typical setup, this fixed number is 2N + 5, with the three types of
moves averaging N, 5, and N, respectively. At the end of each cycle,
the densities of the two systems are calculated and saved. The
average densities in the second half of the simulations are then
taken as those of the two phases (Fig. 5b). By repeating the simula-
tions at different temperatures, the binodal is obtained (Fig. 5c).
2.5 Slab-Geometry Slab-geometry simulations [11] predated Gibbs-ensemble Monte

Molecular Dynamics Carlo and are still growing in popularity [3, 4, 22–25]. Although
Simulations these simulations can be done via Monte Carlo, implementation by
molecular dynamics allows them to be applied to systems modeled
at different levels of details, from point particles to proteins in
explicit solvent. In a typical setup, a dense phase is prepared in a
cubic box (with side length L), for example, by compression
(Fig. 6a top row). Along the +z and –z directions, volumes are
then added, which are either empty space in the case of particle
systems or pure solvent in the case of proteins in explicit solvent
(Fig. 6a middle row). Upon equilibration at constant N, V, and T,
Fig. 5 Illustration of Gibbs-ensemble Monte Carlo. Two separated systems are simulated, with the tempera-
ture, total particle number, and total volume held constant. (a) Three types of Monte Carlo moves: particle
displacement; volume exchange; and particle swap. (b) The densities of the two systems during the
simulations at N ¼ 2048 and T ¼ 1.05; the average densities in the second half of the simulations are
shown as red horizontal lines. Snapshots of the two systems at the end of 100,000 Monte Carlo (MC) cycles
are shown. Each MC cycle consists of, on average, N particle displacements, 5 volume exchanges, and
N particle swaps. (c) The binodal, constructed from the average densities of the two systems at different
temperatures
the additional volumes become the dilute phase, in coexistence

with the dense phase in the middle (Fig. 6a bottom row).
2.5.1 Calculating and Once the system reaches equilibration, snapshots are saved for
Fitting the Density Profile analysis. The density profile along z is obtained by dividing the
simulations box into thin slices, each with cross section L2 and
thickness Δz (Fig. 6b). The number of particles in each slice is
counted to yield the density. The density profile is then averaged
over the saved snapshots. Finally, one half of the average density
profile is fit to a hyperbolic tangent function (Fig. 6c):
ρden þ ρdil ρden ρdil z z0
ρðz Þ ¼ þ tanh ð11Þ
2 2 d
where ρden and ρdil denote the density of the dense and dilute
phases, respectively, and z0 and d denote the central position and
width, respectively, of the interface between the phases.
Fig. 6 Setup and data analysis of slab-geometry simulations. (a) The system, initially in a cubic box, is
compressed in all directions to reach a density around 0.7. The smaller cubic box, with side length L, is then
padded in the +z and –z directions with empty rectangular boxes that each have length 2L. Finally, the system
is allowed to equilibrate in molecular dynamics simulations at constant N, V, and T. (b) Calculation of density
profile along z. The simulation box (at N ¼ 20,000 and T ¼ 0.85) is divided into slices, with cross section L2
and thickness Δz. The number, ni, of particle in slice i is counted to yield the density ρi ¼ ni/(L2Δz). (c) The
density profile (blue curve), averaged over saved snapshots, and its fit to a hyperbolic tangent function (red).
(d) Contribution of a pair interaction to the z-dependent pressure tensor, according to Irving and Kirkwood
[33]. For every pair of particles (i and j) within the cutoff distance, the number (ns) of slices with thickness Δz
that span the displacement, zij ¼ zj – zi, is counted. The contribution of this pair to the pressure tensor
(Eq. (15)) is then distributed equally among the ns slices. Finally, within each slice, the pieces from all the
interaction pairs are summed to yield the z-dependent pressure tensor
2.5.2 Calculating the The interfacial tension γ can be calculated by two methods. Accord-
Interfacial Tension ing to Kirkwood and Buff [32], the expression for γ is
Lz pxx þ pyy
γ¼ pzz ð12Þ
2 2
where the first factor of 2 accounts for the fact that there are two
interfaces in the simulation box, Lz denotes the length of the
simulation box in the z direction, and pαβ are elements of the
pressure tensor. The latter is given by
* +
1 X X
N N 1
N kB T
pαβ ¼ δαβ þ r f ð13Þ
V V i¼1 j ¼1 ij α ij α
where δαβ ¼ 1 when α ¼ β and 0 otherwise, rijα is the α component

of the interparticle displacement vector rij, and fijα is the α compo-
nent of the force fij on particle i exerted by particle j (Fig. 6d).
The Irving-Kirkwood method [33] finds the interfacial tension
from the position-dependent pressure tensor:
Z
1 L z =2 pxx ðz Þ þ pyy ðz Þ
γ¼ dz pzz ðz Þ ð14Þ
2 L z =2 2
The z-dependent pressure tensor is given by

1
pαβ ðz Þ ¼ ρðz ÞkB T δαβ þ 2
L
* +
XN N X 1
1 z zi zj z
r ij α f ij α Θ Θ ð15Þ
z ij z ij z ij
i¼1 j ¼1
where Θ(x) ¼ 1 if x 0 and 0 otherwise. The product of the two Θ

functions serves to select ij pairs for the summation only if z lies
between zi and zj. For each such pair, the number of slices, ns, with
thickness Δz that span zij is counted; the contribution of this ij pair,
rijαfijα/ j zijj, is distributed equally to the ns slices (Fig. 6d).
2.6 Fitting Binodals To facilitate the analysis of system size dependence and the com-
to the Law of parison across methods, we fit the binodals to known functions.
Rectilinear Diameters The mean density of the two phases is fit to the law of rectilinear
diameters [34]
ρden þ ρdil
¼ ρc þ AðT T c Þ ð16Þ
2
and simultaneously the difference in density is fit to a scaling law
ρden ρdil
¼ B ðT c T Þb ð17Þ
2
In these expressions, ρc is the critical density, b is a critical exponent
set to 0.32, and A and B are constants.
2.7 Correction for To correct for finite system size, we obtain binodals for a range of
Finite System Sizes sizes and look for scaling relations between each of the four para-
meters (e.g., Tc) and system size. For FMAP, size dependence is
produced by increasing the volume (or, equivalently, the side length
L of the cubic box) of the systems. The critical temperature is fit to
the following relation:
T c ¼ T c1 þ CL 2 ð18Þ
where Tc1 is the critical temperature extrapolated to infinite system
size, and C is a constant. The other three parameters, ρc, A, and B,
do not have a significant dependence on size, and we simply use the
average values among the different sizes as the values appropriate
for infinite size. None of the four parameters obtained by Gibbs-

ensemble Monte Carlo has a significant dependence on system size,
when the number of particles is at least 256. We again use average
values among different system sizes (excluding N ¼ 128) as those
for infinite size.
Results from slab-geometry molecular dynamics simulations
have the strongest dependence on system size. Each of the four
parameters is fit to the following relation:
q ¼ q 1 þ CN 1 ð19Þ
The interfacial tension at a given temperature is fit to a similar
relation
γ ¼ γ 1 þ DN 2=3 ð20Þ
3 Implementations
Here we present the procedures for obtaining the binodals of a

Lennard-Jones fluid from three routes. Computer codes and details
are provided so that the reader can carry out these simulations and
calculations on their own.
3.1 Chemical- Excess chemical potentials are calculated from configurations gen-
Potential-Based Route erated from Monte Carlo simulations at constant N, V, and T, by
either brute-force particle insertion or FMAP-enabled insertion.
These codes are available at https://github.com/hzhou43/
MiMB_simulations/tree/main/FMAP. The FFT in our FMAP
method for calculating chemical potentials uses the FFTW package
(https://www.fftw.org/).
The fit of the chemical potential to a 5th-order polynomial
function of particle density (Fig. 4b) is modeled after polysolve
(https://arachnoid.com/polysolve/). The intersections of a hori-
zontal line with the van der Waals loop (Fig. 4c) are determined by
the Newton-Raphson method. The areas above and below this line
are obtained by numerical integration using the midpoint rule. The
chemical potential that bisects the van der Waals loop with equal
areas above and below is again found by the Newton-Raphson
method. These steps are implemented into a web server at
https://pipe.rcc.fsu.edu/LLPS/.
To run their own simulations and generate the results pre-
sented in Figs. 7 and 8a top, all the reader needs is a web browser
that supports JavaScript and WebAssembly (https://webassembly.
org/), which enables near-native code execution speed in the web
browser. Through WebAssembly, the reader runs Monte Carlo
simulations of the Lennard-Jones fluid and carries out brute-force
or FMAP-enabled particle insertions on their own browser, com-
pleting a single excess chemical potential calculation in as little as a
few minutes.
Fig. 7 Comparison of binodals calculated from chemical potentials determined by FMAP and by brute-force
insertion. The volumes of the systems at different densities are fixed at L3 with L ¼ 8
Fig. 8 Dependence of FMAP binodals on system size. (a) Top: binodals at three system sizes. Relative to the
series of systems at L ¼ 8, the particle numbers are doubled and quadrupled, respectively; the volume is
increased by nearly the same factors, with L increasing to 10 and 12.8. The grid spacing is fixed at 0.1. The
inset shows a zoom into the region near the critical point, where size effect is most prominent. Note the
narrowing of the binodals with increasing system size. Bottom: fit of the binodal at L ¼ 12.8 to Eqs. (16)
and (17). (b) Effects of system size on the fitting parameters. The critical temperature decreases with
increasing L as Tc1 + C/L2 (curve, with Tc1 ¼ 1.17 and C ¼ 0.9), consistent with the narrowing of the
binodals, but the other three parameters lack any trend, as indicated by the comparison against a
horizontal line
3.1.1 Obtaining Chemical 1. From a web browser that supports JavaScript and WebAssem-
Potential Through Monte bly, go to
Carlo (MC) Simulation and https://pipe.rcc.fsu.edu/LLPS/mclj/
Brute-Force Insertion (Fig.
2. Set input parameters for simulations. Default values are as
2)
follows: number of particles (N), 30; box side length (L), 8.0
(see Note 3); temperature (T), 0.65; number of MC steps for
equilibration, 2,000,000; number of MC steps for production,
20,000,000. Here an MC step is an attempted displacement of
one particle. An entry for random number seed is also provided
in case the reader wants to check reproducibility using different
random number seeds.
3. Set input parameters for brute-force insertions. Default values
are as follows: number of MC steps between insertions,
10,000; number of insertions per snapshot, 10,000.
4. Click run to obtain the excess chemical potential for one N, or
one particle density, N/V.
5. Repeat steps 2–4 with N set to 30i, where i goes from 2 to 15.
6. Repeat steps 2–5 by changing the temperature, covering
0.65–1.15.
3.1.2 Obtaining Chemical 1. From a web browser that supports JavaScript and WebAssem-
Potential Through MC bly, go to
Simulation and FMAP- https://pipe.rcc.fsu.edu/LLPS/mcljfft/
Enabled Insertion (Fig. 3)
2. Set input parameters for simulations as in step 2 in
Subheading 3.1.1.
3. Set input parameters for FMAP-enabled insertions. Default
value is number of MC steps between insertions, 10,000.
Note that FMAP is equivalent to uniform insertions in the
simulation box. With L ¼ 8 and a grid spacing of 0.1, there
are a total of 803 ¼ 512,000 grid points, and hence, one FMAP
calculation equals 512,000 brute-force insertions. When the
volume is doubled or quadrupled, the total number of grid
points increases by the same factors (with grid spacing fixed at
0.1).
4. Click run to obtain the excess chemical potential for one parti-
cle density.
5. Repeat steps 2–4 with N set to 30i, where i goes from 2 to 15.
6. Repeat steps 2–5 by changing the temperature, covering
0.65–1.15.
7. Repeat steps 2–6 by doubling the volume and particle num-
bers, with L increased to 10.0 and N increased to 60i, i ¼ 1–15.
8. Repeat steps 2–6 by quadrupling the volume and particle
numbers, with L increased to 12.8 and N increased to
120i, i ¼ 1–15.
3.1.3 Constructing 1. From a web browser that supports JavaScript, go to:

Binodals from Chemical https://pipe.rcc.fsu.edu/LLPS/
Potential Data
2. Copy the precomputed chemical potential data from brute-
force insertions at L ¼ 8 to the “Number Density and Excess
Chemical Potential Data Entry Area.” Enter the data as two
columns: the first column is particle density (e.g., particle
number divided by volume), and the second column is excess
chemical potential in units of kBT.
3. Inspect the dependence of the excess chemical potential on the
particle density. Fit the points to a 5th-order polynomial with-
out a constant term (Fig. 4b). Check the fit quality and the
polynomial coefficients.
4. Add the ideal part to the polynomial fit to obtain the full
chemical potential.
5. Set an initial chemical potential to find the coexistence line
through an equal-area construction (Fig. 4c). Iterate using
the Newton-Raphson method. The numerical results can be
verified against WolframAlpha (https://www.wolframalpha.
com/). This step finds two points on the binodal at the given
temperature and two points on the spinodal at the same
temperature.
6. Repeat steps 2–5 with data at different temperatures to obtain
the binodal and spinodal for a range of temperatures.
7. Repeat steps 2–6 using chemical potential data obtained from
FMAP at L ¼ 8. Compare the binodals from the two methods
(Fig. 7).
8. Repeat step 7 to obtain the binodal using FMAP data at
L ¼ 10.0.
9. Repeat step 7 to obtain the binodal using FMAP data at
L ¼ 12.8.
10. Comparing results from steps 7–9 to check the dependence of
FMAP binodals on system size (Fig. 8 top).
3.1.4 Dependence on As shown in Fig. 8a top, when the system size increases, the
and Correction for System binodals narrow slightly near the critical point. To quantify the
Size effects of system size, we fit the binodals to the law of rectilinear
diameters (Eq. 16) and the scaling law of Eq. (17) (Fig. 8a bot-
tom). In Fig. 8b, we display the dependence of the four fitting
parameters, Tc, ρc, A, and B, on system size. Consistent with the
narrowing of the binodals, Tc decreases with increasing L. A fit to
the scaling relation of Eq. (18) yields a value of 1.17 for the critical
temperature, Tc1, when the system size is extrapolated to infinity.
The other three parameters have no clear dependence on system
size and show only small deviations from a mean value.
Fig. 9 Details of the three types of Monte Carlo moves. Each Monte Carlo cycle consists of a total of 2 N + 5
moves, distributed among the three types with probabilities of N/(2 N + 5), 5/(2 N + 5), and N/(2 N + 5),
respectively. (a) For each particle displacement, a particle is randomly selected from the list of N. In the case
illustrated, the selected particle is in system I. A displacement is then proposed, with uniform probability
within a cube centered at the original position and with side length 2lmax. The would-be change in the total
energy, ΔUI, of system I is calculated, and the proposed displacement is accepted with the probability shown,
where ΔUII ¼ 0. (b) For each volume exchange, the volume of system I is proposed to increase by ΔV, where
ΔV is a random value picked between –ΔVmax and ΔVmax with uniform probability, and the volume of system II
is proposed to decrease by ΔV. In the case illustrated, ΔV is negative. The proposed volume exchange is
accepted with the probability shown. (c) For each particle swap, the system to which a particle is to be
inserted is selected between I and II with equal probability. In the case illustrated, system I is selected for
particle insertion, while system II is chosen for particle removal. The proposed particle swap is accepted with
the probability shown
3.2 Gibbs-Ensemble In these simulations, we prepare two cubic boxes that initially have
Monte Carlo the same volume and the same number of particles, with a density
(ρ0) in the unstable region. For Lennard-Jones particles, a density
of ~0.3 is appropriate. The simulation ensues with three types of
moves: particle displacement, volume exchange, and particle swap
(Fig. 9), leading to one box that contains the dilute phase and one
box that contains the dense phase (Fig. 5b).
3.2.1 Running the We wrote a C++ code, gibbs.cpp, to run the simulations and a
Simulations script, densities.sh, to analyze the trajectory. Both are depos-
ited at https://github.com/hzhou43/MiMB_simulations/tree/
main/GEMC. The input to gibbs.cpp consists of the total parti-
cle number N, the temperature T, and the initial density ρ0. The
command
./gibbs 2048 1.05 0.3
will run the simulations with N ¼ 2048, T ¼ 1.05, and ρ0 ¼ 0.3. It

produces an output file, output.txt, that contains thermody-
namic quantities of interest after each MC cycle (i.e., one line per
MC cycle). Each line reports the total energies UI and UII, volumes
VI and VII, and densities ρI and ρII of the two boxes. In addition,
the code produces a trajectory file (Trajectory.xyz) that con-
tains the particle positions after each MC cycle. The analysis part is
very simple: densities.sh does an ensemble average of the
densities using output.txt as input, to produce the densities,
ρdil and ρden, of the two phases. Below we break the gibbs.cpp
code into different parts. The two boxes are referred to as 0 and 1 in
the code. Actual lines of code are written in Arial font, for example,
initialize();
pseudo code is additionally in italic, for example,
energy(0);
3.2.2 System Setup and 1. Initialize the systems with the function
Simulation
initialize();
which places half of the N particles on a cubic lattice in each

box. The side length of the boxes is L ¼ (N/2ρ0)1/3.
2. Calculate the initial energy of each box (see Note 4):
energy(0);
energy(1);
3. Now start the simulation. Each MC cycle consists of pdisp

+vexch+pswap moves, with pdisp, vexch, and pswap
assigned values of N, 5, and N, respectively. On average, the
numbers of particle displacements, volume exchanges, and
particle swaps are pdisp, vexch, and pswap, respectively.
For each MC move, its type is determined by the random
integer
des = (rand()/double(RAND_MAX))*(pdisp+vexch+pswap);
4. If particle displacement is selected (Fig. 9 top row; see Note 5),

go to
particle_disp();
where a particle is randomly selected from the list of N:
p = int(N*(rand()/double(RAND_MAX)));
Propose a particle displacement in the interval (-lmax, lmax)

in each direction:
xd = x[p] + (2*(rand()/double(RAND_MAX)) - 1)*lmax;

yd = y[p] + (2*(rand()/double(RAND_MAX)) - 1)*lmax;
zd = z[p] + (2*(rand()/double(RAND_MAX)) - 1)*lmax;
and calculate the resulting energy change:
de = ener();
which basically calculates the interaction energies of the pro-

posed particle with the rest of the system in the same box, once
before the displacement and once after the displacement. Apply
Metropolis criterion for the proposed displacement:
ran= rand()/double(RAND_MAX);
if (ran < exp(-beta*de)){
x[p] = xd;
y[p] = yd;
z[p] = zd;
}
5. If volume exchange is selected (Fig. 9 middle row), go to
volume_exch();
Calculate the energy (see Note 6) and volume of each box:
eno[0] = energy(0);
eno[1] = energy(1);
vo[0] = pow(L0[0],3.0);
vo[1] = pow(L0[1],3.0);
Propose a volume change in the (-dVmax, dVmax) range:
vn[0] = vo[0]+(2*(rand()/double(RAND_MAX)) - 1)*dVmax;

vn[1] = Vn-vn[0]; //Vn total volume of the 2 boxes (conserved)
Scale all particle positions by a factor:
fact[i]=Ln[i]/Lo[i]; //for i=0, 1

for(int i=0; i<N; i++){
xv[i] = x[i]*fact[boxId[i]]; //boxId[i]=0 or

1 (box that contains particle i)
yv[i] = y[i]*fact[boxId[i]];
zv[i] = z[i]*fact[boxId[i]];
}
Calculate the energies of the boxes after the proposed move:
enn[0] = energy(0);
enn[1] = energy(1);
Apply Metropolis criterion for this move:
arg1 = -beta*(enn[0]-eno[0])+N[0]*log(vn[0]/vo[0]);
arg2 = -beta*(enn[1]-eno[1])+N[1]*log(vn[1]/vo[1]);
if (ran < exp(arg1+arg2)){ //accept the move
L[0] = pow(vn[0],1.0/3.0);
L[1] = pow(vn[1],1.0/3.0);
for(int i=0; i<N; i++){
x[i] = xv[i];
y[i] = yv[i];
z[i] = zv[i];
}
}
6. If particle swap is selected (Fig. 9 bottom row; see Note 7), go

to
particle_swap();
Randomly pick a box for particle insertion and the other box
for particle removal:
p = rand()/double(RAND_MAX);
if (p < 0.5){
add = 0; //add to box0
del = 1; //delete from box1
} else
{. . . //the opposite
}
Pick a random position in the “add” box for the particle to be

inserted:
xa = (rand()/double(RAND_MAX))*L[add];
ya = (rand()/double(RAND_MAX))*L[add];
za = (rand()/double(RAND_MAX))*L[add];
Pick a particle from the “del” box for removal:
do{
pd = int((rand()/double(RAND_MAX))*N); //
particle ID
}while(boxId[pd]!=del);
Calculate the interaction energy of the removed particle and

the rest of the “del” box, and the interaction energy of the
inserted particle with the rest of the “add” box. Obtain the
energy change:
de = ener(add)-ener(del);
Now apply Metropolis criterion for the proposed move:
arg = -beta*de+log(v[add]/(Np[add]+1)*Np[del]/v
[del]);
if (ran < exp(arg)) {//accept the move
Np[add] += 1;
Np[del] -= 1;
boxId[pd] = add;
x[pd] = xa;
y[pd] = za;
z[pd] = za;
}
7. Repeat steps 3–6 for 100,000 cycles.
3.2.3 Analyzing 1. Plot the densities of the two boxes reported by the output file,
output.txt and output.txt (Fig. 5b). After a few thousand MC cycles, the
Constructing Binodals densities should plateau. Run the script
./densities.sh
to obtain the average densities, ρdil and ρden, in the second half
of the 100,000 MC cycles.
2. Repeat Subheading 3.2.2 and the preceding step for a range of
temperatures. Plot ρdil and ρden as a function of T to produce
the binodal at N ¼ 2048 (Fig. 5c).
3. Repeat Subheading 3.2.2 and the preceding two steps for
N ¼ 1024, 512, 256, and 128, to check for possible effects of
system size. As shown in Fig. 10a top, except for the smallest
system size (N ¼ 128), the binodals agree with each other
rather well. Therefore, starting from N ¼ 256, the binodals
Fig. 10 Weak dependence of GEMC binodals on system size. (a) Top: binodals at five system sizes. The
average density of the systems of the GEMC simulations is fixed at 0.3, while the total number of particles
increases from 128 to 2048. The binodals at the four larger sizes essentially agree with each other, but that at
N ¼ 128 shows a minute leftward shift (toward lower densities), especially in the dense-phase branch. (b)
Bottom: fit of the binodal at N ¼ 2048 to the law of rectilinear diameters. (b) Effects of system size on the
fitting parameters. The values of each parameter at the different system sizes are very close to each other,
except for a slightly lower ρc and a slightly lower B at N ¼ 128, corresponding to the minute leftward shift and
narrowing of the binodal
calculated by Gibbs-ensemble Monte Carlo become size

independent.
4. Fit the binodals as described in Subheading 3.1.4 (Fig. 10a
bottom). The values of the four fitting parameters verify the
size independence (Fig. 10c). For each parameter, the values at
different system sizes show only small fluctuations around a
mean value, except for a slightly lower ρc and a slightly lower
B at N ¼ 128. The mean Tc is 1.170.
3.3 Slab-Geometry This method works as illustrated in Fig. 6a. We start by placing
Molecular Dynamics particles in a cubic box at a low initial density, and then compress
Simulations the box till the density reaches ~0.7 (with side length of the cubic
box at L). Next we elongate the box in +z and –z directions to a
length of Lz ¼ 5L, creating a dense slab in the middle of the
simulation box. Finally molecular dynamics simulations equilibrate
the system to the phase-separated state with the dense phase in the
middle and the dilute phase on the two sides.
3.3.1 Temperature Although slab-geometry simulations can be done via Monte Carlo,
Regulation implementation by molecular dynamics has much wider applicabil-
ity, and is done here. In Monte Carlo, one only needs to specify the
Fig. 11 Illustration of a time step in Langevin dynamics simulations. (a) Motions

of particles in one time step. (b) Governing equations of particle motions. (c)
Algorithm for advancing particle positions and velocities in one time step.
Symbols are as follows: Fi, force on particle i due to interparticle interactions;
ζ, damping constant; Ri and Riα, random force and its Cartesian component; δt,
time step; Gi and Gi0 , 3-dimensional vectors with components given by random
variables with zero mean and unit standard deviation
temperature of the system and use it to control the probabilities of

accepting proposed moves (see Figs. 2b and 9). In molecular
dynamics simulations, particles move step by step according to
the force exerted on them (Fig. 11a). To run these simulations at
a constant temperature, one has to specifically regulate the temper-
ature of the system to the desired value. Various thermostats have
been introduced, by coupling the system to a thermal bath and
thereby modifying the original equations of motion. Here we use
the Langevin thermostat, whereby the equations of motion are
modified into the Langevin equation, including a frictional force
and a random force (Fig. 11b). Correspondingly, the algorithms for
advancing the particle positions and velocities have to be modified.
Figure 11c presents the velocity Verlet algorithm.
The modification of the equations of motion needed for tem-
perature regulation perturbs the dynamics of the system. Correc-
tions for such perturbations have been found for the Langevin
thermostat [35]. Here we only deal with thermodynamic proper-
ties, and hence no need for such corrections.
3.3.2 Overall Description All simulations are performed at constant N, V, and T using
HOOMD-Blue (version 2.5.0) on GPUs, with a user-friendly
Python interface [36]. The setup, simulation, and analysis are all
driven by a script named submit.pbs, which contains the follow-
ing lines:
1. python3 init.py
# Randomly place N particles into a large box to create a dilute
state
2. python3 compression.py
# Compress the system to achieve a density of ~0.7
3. python3 elong.py
# Elongate the compressed cubic box to add empty space on
the two sides
4. python3 equilibration.py --user¼“0.85”
# Run simulation at T ¼ 0.85; generate log file and trajectory
file
5. python3 coord.py -- user ¼ “0.85”
# Extract particle (x, y, z) coordinates from trajectory file into
text file Tcoor.txt
6. ./ik 0.85 ns Mstart
# Calculate density and pressure profiles with Tcoor.txt as
input (ns ¼ Lz/Δz; Mstart, starting snapshot for data
analysis)
7. gnuplot fit.gnu
# Fit density profile to a hyperbolic tangent function

8. gnuplot binodal.gnu
# Fit binodal
9. ./surface_tension.sh
# Calculate KB and IK surface tension values, with log file and
pressure profile as input
The submit.pbs file, Python scripts for running HOOMD, C
++ code ik.cpp, gnuplot scripts, and shell script surface_-
tension.sh are all deposited at https://github.com/
hzhou43/MiMB_simulations/tree/main/SLAB. Next we
present details for each of the nine script files.
3.3.3 init.py This script carries out the following steps in HOOMD (Fig. 6a).
1. Create N ¼ 20,000 particles (i.e., polymers with one bead):
polymer=dict(bond_len=1.2,type=[‘A’]*1,bond=”linear”,
count=20000)
2. Randomly place the particles into a cubic box of side length

60 to achieve a dilute system:
system=hoomd.deprecated.init.create_random_polymers(
box=hoomd.data.boxdim(L=60),
polymers=[polymer1],separation=dict(A=0.25),
seed=52)
3. Create a neighbor cell list:
nl=md.nlist.cell(check_period=1)
4. Define the Lennard-Jones shifted potential with cutoff distance

rcut ¼ 3.0:
lj = md.pair.lj(r_cut=3.0,nlist=nl)
5. Define the well depth (ε) and contact distance (σ):
lj.pair.coeff.set(‘A’,’A’,epsilon=1.0, sigma=1.0)
6. Group all particles present into the same class:
all = hoomd.group.all()
7. Set parameters for energy minimization:
minimize.md.integrate.mode_minimize_fire(. . .)
8. Specify thermodynamic quantities to be saved in a log file:
hoomd.analyze.log(
‘log-output_init.log’,quantities=[‘tempera-
ture’,’potential_energy’], period=10)
9. Set parameters for the trajectory file:
hoomd.dump.gsd(‘init.gsd’,period=1000, group=all, overwri-

te=True)
10. Run the minimization for 5000 steps:
hoomd.run(5000)
3.3.4 compression.py This script creates the initial dense slab of ρ ~ 0.7 by compressing
the box side length from 60 to 30 (Fig. 6a). The input is the last
snapshot from the init.gsd of step 10 in Subheading 3.3.3.
1. Set up the system:
system=hoomd.init.read_gsd(‘init.gsd’,frame=-1)
2. Set initial and final side length for compression:
initial_L=60
final_L=30
shrink_L=hoomd.variant.linear_interp([(0,
initial_L), (5000,final_L)])
resize=hoomd.update.box.resize(L=shrink_L,
period=1, scale_particle=True)
3. Define the interaction potential and particles as in steps 3–6 in

Subheading 3.3.3.
4. Set the integration time step:
hoomd.md.integrate.mode_standard(dt=0.005)
5. Select the Langevin thermostat and set parameters:
kT=4.0
integrator=hoomd.md.integrate.langevin(group=all,
kT=kT, seed=42)
integrator.set_gamma(‘A’,gamma=0.1)
6. Set the log file as log-output_compress.log and trajectory

file as compress.gsd, as in steps 8 and 9 in Subheading 3.3.3.
7. Run for 20,000 time steps:
hoomd.run(20000)
3.3.5 elong.py This script, running outside HOOMD, changes Lz from L to 5L

(Fig. 6a; see Note 8). The snapshots before and after box elonga-
tion are saved in elong.gsd.
3.3.6 equili- This script equilibrates the elongated system prepared in Subhead-
bration.py – ing 3.3.5, at T ¼ 0.85.
user¼“0.85”
1. Set up the system:
system=hoomd.init.read_gsd(‘elong.gsd’,frame=-1)
2. Select the shifted Lennard-Jones potential (Eq. (2)):
lj = md.pair.lj(r_cut=3.0,nlist=nl)
lj.pair.coeff.set(‘A’,’A’,epsilon=1.0, sigma=1.0)
lj.set_params(mode=“shift”)
3. Specify the integration time step as in step 4 in

Subheading 3.3.4.
4. Specify the thermostat as in step 5 in Subheading 3.3.4, except
with kT¼0.85.
5. Set the log file as log-output_equilT0.85.log and trajec-
tory file equil_T0.85.gsd.
6. Run for 10,000,000 time steps:
hoomd.run(10e7)
3.3.7 Data Analysis at a We use only the second half of the simulations in Subheading 3.3.6
Single Temperature for data analysis. This means the second 500,000 lines of log-
output_equilT0.85.log and the second 5000 snapshots in
equil_T0.85.gsd. The analysis yields the densities of the dilute
and dense phases as well as the interfacial tension values determined
by the Kirkwood-Buff and Irving-Kirkwood methods.
1. With equil_T0.85.gsd as input, coord.py extracts the
(x, y, z) coordinates of the particles and saved them to
Tcoor.txt.
2. With Tcoor.txt as input, ik.cpp calculates the density pro-

file (Fig. 6b, c) and the pressure profile, that is,
pzz(z) [pxx(z) + pyy(z)]/2 (see Eq. (14); see Note 9). The
spacing, Δz, in z is set to 0.1. Because the dense slab can drift
during the simulations, it is important to first place the center
of mass of the system in each snapshot to the origin before
averaging over the snapshots.
3. With the density profile as input, fit.gnu does a fit in gnu-
plot to a hyperbolic function (Eq. (11); Fig. 6c) to yield the
densities of the dilute and dense phases at the present
temperature.
4. surface_tension.sh calculates the interfacial tension
according to both the Kirkwood-Buff (KB) and the Irving-
Kirkwood (IK) method. For KB, the input is the diagonal
elements of the pressure tensor saved in the .log file; for IK,
the input is the pressure profile calculated in step 2.
3.3.8 Constructing 1. Repeat Subheadings 3.3.6 and 3.3.7 for a range of

Binodals and Analyzing temperatures.
Dependence on System 2. Use binodal.gnu to plot the densities of the two phases at
Size different temperatures as a binodal (Fig. 12a top), and to fit the
binodal to Eqs. (16) and (17) (Fig. 12a bottom).
3. Repeat steps 1–2 for other particles numbers.
4. Plot the binodal fitting parameters as a function of N, and fit to
the scaling relation of Eq. (19) (Fig. 12b).
Fig. 12 Dependence of slab-geometry binodals on system size. (a) Top: binodals at six system sizes. Both
N and L increase, but the density N/L3 is kept around 0.7. With increasing system size, the binodals broaden
near the critical point. Bottom: fit of the binodal at N ¼ 50,000 to the law of rectilinear diameters. (b)
Dependences of the fitting parameters on system size, modeled by the function q ¼ q1 + C/N. For Tc, ρc, B,
and A, the values of (q1, C) are as follows: (1.175, 43.1), (0.316, 16.0), (0.511, 19.8), and (0.190,
58.5), respectively
3.3.9 Dependences of 1. Plot the pressure profiles and its integrations over z for differ-
Interfacial Tension on ent temperatures at N ¼ 20,000 (Fig. 13a).
Temperature and System 2. Compare the KB and IK results (Fig. 13b). As expected, excel-
Size lent agreement is seen.
3. Plot the KB results as a function of temperature for different
N values (Fig. 14a). Modest dependence in system size is
observed (Fig. 14a inset).
4. For each temperature, fit the KB results to the scaling relation
of Eq. (20) (Fig. 14b–d).
4 Concluding Remarks
The binodals calculated by the three methods show different

extents of dependence on system size. Gibbs-ensemble Monte
Carlo has the weakest size dependence (Fig. 10). Starting from
N ¼ 256, the binodals are essentially size independent. FMAP has
an intermediate size dependence (Fig. 8). When the binodals are fit
to Eqs. (16) and (17), only one of the four fitting parameters, Tc,
has some size dependence, and a scaling relation allows us to find its
value, Tc1, when the system size is extrapolated to infinity. By
comparison, slab-geometry molecular dynamics simulations have
the strongest dependence on system size (Fig. 12). This outcome is
Fig. 13 Interfacial tension results from slab-geometry simulations at N ¼ 20,000. (a) The pressure profile
according to the Irving-Kirkwood method (solid curves), and its integration that yields the interfacial tension
(dashed curves). (b) Comparison of results by the Kirkwood-Buff method (symbols) and by the Irving-Kirkwood
method (lines)
Fig. 14 Dependence of interfacial tension on system size. (a) Interfacial tension results calculated at four
system sizes over a range of temperature. The inset shows a zoom into the data at T ¼ 0.85. (b) Dependence
of interfacial tension at T ¼ 0.65 on system size, modeled by the function γ ¼ γ1 + D/N2/3, with (γ1,
D) ¼ (0.856, 0.801). (c) Corresponding data at T ¼ 0.85, with (γ1, D) ¼ (0.468, 0.767). (d) Corresponding
data at T ¼ 0.95, with (γ1, D) ¼ (0.292, 0.606)
likely related to the fact that interfaces are present in these simula-
tion systems. Again, by running simulations up to very large system
sizes (i.e., 50,000 particles) and using a scaling relation, we can find
the values of the four binodal parameters when the system size is
extrapolated to infinity.
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Phase Separated Biomolecular

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For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

This volume on phase-separated biomolecular condensates comes at an opportune time. As

Chapter Methods Applications Unique angle

Chapter Methods Applications Unique angle

concentrations and diffusion constants of condensate components. The former focuses on

Chicago, IL, USA Huan-Xiang Zhou

12 Determining Thermodynamic and Material Properties of Biomolecular

IBRAHEEM ALSHAREEDAH • Department of Physics, University at Buffalo SUNY, Buffalo, NY,

JINGE GU • Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute

MELISSA D. STUCHELL-BRERETON • Department of Biochemistry and Molecular Biophysics,

Calculating Binodals and Interfacial Tension

Phase-separated biomolecular condensates are revolutionizing our

experimental verification and led to the classification of all macro-

All molecular simulations can only be done on a system size far

We illustrate the three methods for calculating binodals on a

Fig. 2 Illustration of calculating the chemical potential by Widom insertion. (a)

is the sum of the interaction energies between an inserted particle

u12 ðrÞ ¼ q 1 q 2 ϕðrÞ ð7Þ

where δ(x) is a three-dimensional delta function and g(x) is the

We can recognize the right-hand side of Eq. (8) as the cross-

equivalent to brute-force insertion at all the grid points, but at a

2.3 Maxwell Once the chemical potential at a given temperature is obtained as a

Fig. 4 The excess chemical potential, calculated by FMAP, over a range of

In practice, using the method described in Subheading 2.1 or

extrema define the so-called spinodal. Between these two densities,

2.5 Slab-Geometry Slab-geometry simulations [11] predated Gibbs-ensemble Monte

the additional volumes become the dilute phase, in coexistence

where δαβ ¼ 1 when α ¼ β and 0 otherwise, rijα is the α component

The z-dependent pressure tensor is given by

where Θ(x) ¼ 1 if x 0 and 0 otherwise. The product of the two Θ

for infinite size. None of the four parameters obtained by Gibbs-

Here we present the procedures for obtaining the binodals of a

3.1.3 Constructing 1. From a web browser that supports JavaScript, go to:

./gibbs 2048 1.05 0.3

will run the simulations with N ¼ 2048, T ¼ 1.05, and ρ0 ¼ 0.3. It

pseudo code is additionally in italic, for example,

which places half of the N particles on a cubic lattice in each

3. Now start the simulation. Each MC cycle consists of pdisp

4. If particle displacement is selected (Fig. 9 top row; see Note 5),

where a particle is randomly selected from the list of N:

Propose a particle displacement in the interval (-lmax, lmax)

xd = x[p] + (2*(rand()/double(RAND_MAX)) - 1)*lmax;

and calculate the resulting energy change:

which basically calculates the interaction energies of the pro-

5. If volume exchange is selected (Fig. 9 middle row), go to

Calculate the energy (see Note 6) and volume of each box:

xd = x[p] + (2(rand()/double(RAND_MAX)) - 1)lmax;

vn[0] = vo[0]+(2(rand()/double(RAND_MAX)) - 1)dVmax;