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Crystals
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Membrane Protein Crystallography
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Membrane Protein Crystallography

A special issue of Crystals (ISSN 2073-4352). This special issue belongs to the section "Biomolecular Crystals".

Deadline for manuscript submissions: closed (21 July 2023) | Viewed by 11715

Special Issue Editors

Dr. Qun Liu

E-Mail Website
Guest Editor
1. Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA
2. Photon Science, NSLS-II, Brookhaven National Laboratory, Upton, NY 11973, USA
Interests: membrane protein structure; x-ray crystallography; microcrystals; membrane protein crystallization
Dr. Dianfan Li

E-Mail Website
Guest Editor
CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China
Interests: membrane protein structure and function; lipid cubic phase crystallization; lipid and lipoprotein biogenesis
Dr. Youzhong Guo

E-Mail Website
Guest Editor
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298, USA
Interests: membrane proteins; protein crystallography; protein crystallization
Dr. Albert Guskov

E-Mail Website
Guest Editor
Groningen Biomolecular Sciences and Biotechnology Institute, Faculty of Science & Engineering, University of Groningen, 9747 AG Groningen, The Netherlands
Interests: membrane proteins; protein crystallography; protein crystallization
Special Issues, Collections and Topics in MDPI journals
Dr. Michael C. Wiener

E-Mail Website
Guest Editor
Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22903, USA
Interests: structure/function of integral membrane proteins; structural biophysics; enzymology and virology of ZMPSTE24; sparse-constraint structure determination; technology development

Special Issue Information

Dear Colleagues,

The three-dimensional atomic structures of membrane proteins are indispensable for understanding their molecular mechanisms and provide the structural bases for rational protein engineering and inhibitor design. Although membrane proteins comprise approximately 30% of all proteins in living organisms, only 1.5% of proteins available in the Protein Data Bank (PDB) are membrane protein structures. Crystallography is a powerful technology for protein structure determination. Indeed, since 1985, the first high-resolution atomic structure of a photosynthetic reaction center from the bacterium Rhodopseudomonas virdis was determined using X-ray crystallography; many such structures have since been determined through this method. However, membrane protein structure determination through crystallography presents tremendous challenges due to the limitation of crystallization. Many innovative alternative crystallization strategies have thus been developed.

Recent advances in cryo-EM technology have thrust membrane protein structural biology into the spotlight. Many crystallization-resistant membrane proteins can be investigated by single-particle cryo-EM or cryo-EM tomography (especially large membrane protein complexes). Due to its advantages, single-particle cryo-EM has attracted many structural biologists, mainly crystallographers; however, the further development of membrane protein crystallography should not be neglected. We need a breakthrough in membrane protein crystallization equal to that of the current direct detector in the cryo-EM field. With this aim in mind, it is critical we summarize the past decades' successes and failures (from 1985 to 2022).

This Special Issue of Crystals will provide a forum for discussion on the current state of membrane protein crystallography by collecting contributions related to the structure and function of membrane proteins and related technology relating to subjects including, but not limited to, the following:

  • Membrane protein expression and purification;
  • Membrane protein reconstitution and characterization;
  • Membrane protein engineering;
  • Pre-crystallization screening for membrane proteins;
  • Membrane protein stabilization in detergents, lipids, etc.;
  • Membrane protein crystallization methods and strategies such as lipid cubic phase;
  • Membrane protein crystallization chaperons (antibody, fusion protein);
  • Phasing membrane proteins using ab initio methods and molecular replacement, including AlphaFold;
  • Preparation and manipulation of microcrystals of membrane proteins;
  • Synchrotron X-ray microdiffraction experiments optimized for membrane protein crystals;
  • Application of X-ray free-electron lasers for studying membrane protein structure;
  • Micro-ED experiments for membrane protein crystals;
  • 2D electron crystallography;
  • Technologies for the investigation of membrane protein structures in distinct conformational states;
  • Exploring membrane protein structural dynamics using time-resolved crystallography;
  • Small-molecule drug discovery and development targeting membrane proteins;
  • Structural analysis of membrane protein complexes.

We welcome research on the structure determination of membrane proteins from all protein families using X-ray crystallography and its remaining challenges. This unique collection will be invaluable for membrane protein crystallographers and general membrane protein researchers in academic and industrial environments. Research articles, review articles, or communications articles from researchers who have made significant contributions in the crystal structure determination of a given membrane protein family are welcome.

Dr. Qun Liu
Dr. Dianfan Li
Dr. Youzhong Guo
Dr. Albert Guskov
Dr. Michael C. Wiener
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Crystals is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • membrane protein
  • X-ray crystallography
  • electron crystallography
  • lipid cubic phase
  • detergent
  • receptor
  • channel
  • transporter
  • enzyme
  • drug discovery
  • micro-ED
  • 2D crystal
  • time-resolved crystallography

Published Papers (5 papers)

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Research

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20 pages, 5947 KiB  
Article
Se-MAG Is a Convenient Additive for Experimental Phasing and Structure Determination of Membrane Proteins Crystallised by the Lipid Cubic Phase (In Meso) Method
by Coilín Boland, Chia-Ying Huang, Shiva Shanker Kaki, Meitian Wang, Vincent Olieric and Martin Caffrey
Crystals 2023, 13(9), 1402; https://doi.org/10.3390/cryst13091402 - 21 Sep 2023
Viewed by 856
Abstract
Both intensity and phase information are needed for structure determination by macromolecular X-ray crystallography. The diffraction experiment provides intensities. Phases must be accessed indirectly by molecular replacement, or by experimental phasing. A popular method for crystallising membrane proteins employs a lipid cubic mesophase [...] Read more.
Both intensity and phase information are needed for structure determination by macromolecular X-ray crystallography. The diffraction experiment provides intensities. Phases must be accessed indirectly by molecular replacement, or by experimental phasing. A popular method for crystallising membrane proteins employs a lipid cubic mesophase (the in meso method). Monoolein is the most popular lipid for in meso crystallisation. Invariably, the lipid co-crystallises with the protein recapitulating the biomembrane from whence it came. We reasoned that such a lipid bearing a heavy atom could be used for experimental phasing. In this study, we replaced half the monoolein in the mesophase with a seleno-labelled analogue (Se-MAG), which has a selenium atom in the fatty acyl chain of the lipid. The lipid mixture formed the cubic mesophase and grew crystals by the in meso method of the alginate transporter, AlgE, and the lipoprotein N-acyltransferase, Lnt. Se-MAGs co-crystallised with both proteins and were used to obtain phases for high-resolution structure determination by the selenium single-wavelength anomalous diffraction method. The use of such a mixed lipid system may prove to be a general strategy for the experimental phasing part of crystallographic structure determination of membrane proteins that crystallise via the in meso method. Full article
(This article belongs to the Special Issue Membrane Protein Crystallography)
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13 pages, 2037 KiB  
Article
Crystallographic Characterization of Sodium Ions in a Bacterial Leucine/Sodium Symporter
by Akira Karasawa, Haijiao Liu, Matthias Quick, Wayne A. Hendrickson and Qun Liu
Crystals 2023, 13(2), 183; https://doi.org/10.3390/cryst13020183 - 20 Jan 2023
Viewed by 1696
Abstract
Na+ is the most abundant ion in living organisms and plays essential roles in regulating nutrient uptake, muscle contraction, and neurotransmission. The identification of Na+ in protein structures is crucial for gaining a deeper understanding of protein function in a physiological [...] Read more.
Na+ is the most abundant ion in living organisms and plays essential roles in regulating nutrient uptake, muscle contraction, and neurotransmission. The identification of Na+ in protein structures is crucial for gaining a deeper understanding of protein function in a physiological context. LeuT, a bacterial homolog of the neurotransmitter:sodium symporter family, uses the Na+ gradient to power the uptake of amino acids into cells and has been used as a paradigm for the study of Na+-dependent transport systems. We have devised a low-energy multi-crystal approach for characterizing low-Z (Z ≤ 20) anomalous scattering ions such as Na+, Mg2+, K+, and Ca2+ by combining Bijvoet-difference Fourier syntheses for ion detection and f” refinements for ion speciation. Using the approach, we experimentally identify two Na+ bound near the central leucine binding site in LeuT. Using LeuT microcrystals, we also demonstrate that Na+ may be depleted to study conformational changes in the LeuT transport cycle. Full article
(This article belongs to the Special Issue Membrane Protein Crystallography)
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16 pages, 2646 KiB  
Article
Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase
by Zhipu Luo, Weijie Gu, Yichao Wang, Yannan Tang and Dianfan Li
Crystals 2022, 12(7), 976; https://doi.org/10.3390/cryst12070976 - 13 Jul 2022
Cited by 2 | Viewed by 1768
Abstract
Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to [...] Read more.
Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP. Full article
(This article belongs to the Special Issue Membrane Protein Crystallography)
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Review

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23 pages, 6103 KiB  
Review
Morphological Investigation of Protein Crystals by Atomic Force Microscopy
by Silvia Maria Cristina Rotondi, Giorgia Ailuno, Simone Luca Mattioli, Alessandra Pesce, Ornella Cavalleri and Paolo Canepa
Crystals 2023, 13(7), 1149; https://doi.org/10.3390/cryst13071149 - 24 Jul 2023
Cited by 1 | Viewed by 1755
Abstract
In this review, we discuss the progress in the investigation of macromolecular crystals obtained through the use of atomic force microscopy (AFM), a powerful tool for imaging surfaces and specimens at high resolution. AFM enables the visualization of soft samples at the nanoscale [...] Read more.
In this review, we discuss the progress in the investigation of macromolecular crystals obtained through the use of atomic force microscopy (AFM), a powerful tool for imaging surfaces and specimens at high resolution. AFM enables the visualization of soft samples at the nanoscale and can provide precise visual details over a wide size range, from the molecular level up to hundreds of micrometers. The nonperturbative nature, the ability to scan in a liquid environment, and the lack of need for freezing, fixing, or staining make AFM a well-suited tool for studying fragile samples such as macromolecular crystals. Starting from the first morphological investigations revealing the surface morphology of protein crystals, this review discusses the achievements of AFM in understanding the crystal growth processes, both at the micro- and nanoscale. The capability of AFM to investigate the sample structure at the single molecular level is analyzed considering in-depth the structure of S-layers. Lastly, high-speed atomic force microscopy (HS-AFM) is discussed as the evolution to overcome the limitations of low imaging speed, allowing for the observation of molecular dynamics and weakly adsorbed, diffusing molecules. HS-AFM has provided intuitive views and directly visualized phenomena that were previously described indirectly, answering questions that were challenging to address using other characterization methods. Full article
(This article belongs to the Special Issue Membrane Protein Crystallography)
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13 pages, 1763 KiB  
Review
Protein Fusion Strategies for Membrane Protein Stabilization and Crystal Structure Determination
by Shixuan Liu and Weikai Li
Crystals 2022, 12(8), 1041; https://doi.org/10.3390/cryst12081041 - 27 Jul 2022
Cited by 2 | Viewed by 4970
Abstract
Crystal structures of membrane proteins are highly desired for their use in the mechanistic understanding of their functions and the designing of new drugs. However, obtaining the membrane protein structures is difficult. One way to overcome this challenge is with protein fusion methods, [...] Read more.
Crystal structures of membrane proteins are highly desired for their use in the mechanistic understanding of their functions and the designing of new drugs. However, obtaining the membrane protein structures is difficult. One way to overcome this challenge is with protein fusion methods, which have been successfully used to determine the structures of many membrane proteins, including receptors, enzymes and adhesion molecules. Existing fusion strategies can be categorized into the N or C terminal fusion, the insertion fusion and the termini restraining. The fusions facilitate protein expression, purification, crystallization and phase determination. Successful applications often require further optimization of protein fusion linkers and interactions, whose design can be facilitated by a shared helix strategy and by AlphaFold prediction in the future. Full article
(This article belongs to the Special Issue Membrane Protein Crystallography)
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