Preface

VI Contents

Chapter 9 **Role of X-Ray Crystallography in Structural** 

Soushi Miyazaki and Takahiko Kojima

Chapter 12 **Protein-Noble Gas Interactions Investigated by** 

**Autophagy-Related Proteins 333** 

**and Back to Molecular Biology 349**  Alejandro Giorgetti and Stefano Piccoli

**Part 3 Complimentary Methods 365** 

Chapter 16 **Investigating Macromolecular Complexes**

Cristiano Luis Pinto Oliveira

Chapter 17 **Monitoring Preparation of Derivative** 

Chapter 18 **Complementary Use of NMR to** 

Chapter 10 **Ruthenium(II)-Pyridylamine Complexes**

Chapter 11 **X-Ray Structural Characterization of**

**Part 2 Macromolecules 283**

Noriyuki Ishii

Chapter 14 **Crystallographic Studies on**

Viorel Cîrcu and Marin Micutz

Chapter 13 **Crystallization, Structure and Functional**

Dai Oyama

**Studies of Pyridyl-Ruthenium Complexes 219**

**Having Functional Groups via Amide Linkages 239**

**Cyclometalated Luminescent Pt(II) Complexes 255**

**Crystallography on Three Enzymes - Implication on Anesthesia and Neuroprotection Mechanisms 285**  Nathalie Colloc'h, Guillaume Marassio and Thierry Prangé

**Robustness of Isocitrate Dehydrogenases 309** 

Nobuo N. Noda, Yoshinori Ohsumi and Fuyuhiko Inagaki

**in Solution by Small Angle X-Ray Scattering 367** 

Antonello Merlino, Filomena Sica and Alessandro Vergara

**of Protein Morphological Change in Solution 409**  Shin-ichi Tate, Aiko Imada and Noriaki Hiroguchi

**Protein Crystals** *via* **Raman Microscopy 393**

**X-Ray Crystallography for the Analysis**

Chapter 15 **Knowledge Based Membrane Protein Structure Prediction: From X-Ray Crystallography to Bioinformatics**

Single crystal X-ray crystallography is the most common and easily accessible way to determine the molecular structure of any crystalline material. This method provides two kinds of information which are needed for understanding both single molecule properties and bulk material properties:

1. Molecular Structure - Single Molecules:

Unambiguous and three-dimensional information about the structure of the molecular entities. The data useful here are the bond lengths, bond angles, and torsion angles. These details make this method not only useful for structural determination, confirmation, and analysis of three-dimensional geometry of the molecular entities, but also useful for the Structure-Activity Relationship studies, where the structural details are the basis for understanding the chemical reactivity and stability. These data are also useful for understanding most of the spectroscopic data of materials.

2. Intermolecular Interactions (Packing) - Bulk Material:

All the individual molecules have to come together to form the bulk crystal. How the molecules interact with each other in the crystal provides additional useful information. Various possible intermolecular interactions present in the crystalline bulk can be obtained from the X-ray studies, which are useful for the study of bulk properties as well. These can help explain the observed bulk properties and thereby enable us to make predictions of bulk properties based on structures. These interactions include H-bonding, pi-pi stacking, CH-pi interactions, halogen-halogen interactions, and other donor-acceptor interactions. If there is any phase separation in bipolar molecules, they can also be seen clearly. In addition to providing the details of the molecular arrangement in the bulk, we can also obtain the structure of the empty spaces (void spaces, channels or pores etc). These data are useful in widely varied fields, such as the design of pharmaceuticals, crystal engineering, zeolites for gas separation or catalysis, guest-host materials, sensors, organic magnets, and charge transport.

Single crystal X-ray crystallography has moved a long way from the days I started my research career about a quarter of a century ago. In those days, it took about half a year to complete a structure, due to slow computers and serial data collection by the state-

#### XII Preface

of-the-art automated diffractometers of that time. The recent advances of using the CCD area detectors have reduced the time needed for data collection from days to hours, and also enabled the use of smaller crystals, which were impossible to study before. Also, the presence of supercomputers in everybody's desk/lab and the improved structure processing programs have made the structure determination a very quick task. These advances have made it possible that, with minimal expertise, one can obtain a structure in hours or in one day. When good crystals are available, an experienced crystallographer can determine the structure in as little as an hour. Thus, the current status of instruments, computers, and programs make it possible to obtain the structural details of many more molecules, with smaller crystals, with less expertise, and in much shorter time.

Preface XI

well (such as films or polycrystalline materials, and to some extent in amorphous

When we come to this book on X-ray Crystallography, it is a compilation of recent advances in the structural studies in wide areas. The Chapters are divided into three Sections that deal with Small Molecules, Macromolecules, and Complimentary

Section 1 comprises structural studies of small molecules varying from organic molecules to metal complexes. This Section also includes polymorph studies on drug molecules and database analyses for weak interactions. Chapter 1 deals with the conformational analysis of acetyl anthracenes both by single crystal X-ray studies and theoretical studies. Chapter 2 discusses the structural studies of the less-studied calix[8]arenes with strategies to obtain single crystals in this challenging system. Chapter 3 explores the crystal engineering and co-crystallization aspects on polymorphs with emphasis on pharmacological relevance with examples. Chapters 4 and 5 examine the NH---X and Te---X (X=halogen) weak interactions by meta-analyses

Chapters 6-11 deal with structural studies of metal complexes. Chapter 6 focuses on the complexes of dioxolene (quinone based) ligands with various metals. Chapter 7 discusses the copper(I) complexes of Schiff base ligands whereas Chapter 8 analyses the structures of copper-halide binary dianions (CuX4 and Cu2X6). Chapters 9 and 10 examine the ruthenium complexes with the focus on pyridine based ligands and functionalized pyridylamine based ligands respectively. Chapter 11 discusses the

Section 2 covers the structural studies of proteins, including bioinformatics analysis of membrane proteins. Chapter 12 explores the effects of protein-noble gas interactions by structural analysis of data collected for crystals under various pressures of inert gases, which has implications on anesthesia and neuroprotection. Chapter 13 discusses the structural aspects of isocitrate dehydrogenase family of proteins. Chapter 14 explores the structures of autophagy (intracellular bulk degradation) related proteins. Chapter 15 shows the use of experimental/computational multidisciplinary approach based on structural studies, bioinformatics, modelling and model-guided biology experiments to

In Section 3, SAXS, Raman Microscopy, and NMR methods that compliment single crystal methods for difficult macromolecule systems are discussed. Chapter 16 describes a novel method to study macromolecule colloidal solutions by small angle scattering (SAXS) as a new way to get structural information in native state, especially in comparison to a known structure. Chapter 17 uses Raman microscopy to monitor the chemical modification of derivative proteins, either on the X-ray instrument or off the instrument (in-situ or ex-situ). Chapter 18 provides a novel NMR method (DIORITE based on TROSY) to study the morphological changes of proteins in

structures of cyclometallated luminescent platinum complexes.

obtain the structures and mechanisms of very difficult membrane proteins.

solution by comparing with their solid state structures.

materials and concentrated solutions also).

of Cambridge Structural Database.

Methods.

Though these advances create a large volume of data, too much to handle easily by researchers, there are great programs to help the researchers analyze such voluminous data without getting overloaded. The Cambridge Structural Database, which contains over 500,000 structures, has automated programs to search efficiently and its free Mercury program is great for analyzing individual molecules and their packing so effortlessly. The Inorganic Crystal Structure Database (ICSD), which contains over 130,000 structures, is specifically for inorganic structures. The Protein Data Bank (PDB) is the depository for protein structures with over 76,000 structures.

It is also important to point out that there are a few drawbacks to this method, just like for any other method. The major problem is that one needs to get a good quality single crystal. The minor problems are that the hydrogens are not located accurately, and the structural details are accurate only to the solid state and may possibly deviate in solution or liquid state. As explained below, these are not of any serious concern.

As the technology and instrumentation advance, we are able to deal with smaller and smaller crystals, with only human handling ability making the limit. Since we bought the new instrument a decade ago, the recent advances make it possible to use just onetenth the size of the crystal we need in our 'old' instrument. Though synchrotron radiation sources can provide great data with even smaller crystals, those sources are rare and costly, and not necessary for the most part.

Though the hydrogens can be located more accurately using neutron diffraction, those facilities are fairly rare due to the need for nuclear radiation. However, the lost accuracy is not at all a concern in the vast majority of situations.

The structure of the molecules may deviate to some extent in solution, but the crystal structure shows some of the energetically favored form for the molecules. The structure determined in the crystal will be present in solution, if not 100%, to a significant extent. We have had multiple isomers present in solution and only one isomer in solid, with the crystalline structure being the major isomer in solution, though in one case the solution isomer and crystal isomer were totally different. The intermolecular interactions will provide a statistically significant part of all the possibilities for non-crystalline states as well (such as films or polycrystalline materials, and to some extent in amorphous materials and concentrated solutions also).

X Preface

expertise, and in much shorter time.

of-the-art automated diffractometers of that time. The recent advances of using the CCD area detectors have reduced the time needed for data collection from days to hours, and also enabled the use of smaller crystals, which were impossible to study before. Also, the presence of supercomputers in everybody's desk/lab and the improved structure processing programs have made the structure determination a very quick task. These advances have made it possible that, with minimal expertise, one can obtain a structure in hours or in one day. When good crystals are available, an experienced crystallographer can determine the structure in as little as an hour. Thus, the current status of instruments, computers, and programs make it possible to obtain the structural details of many more molecules, with smaller crystals, with less

Though these advances create a large volume of data, too much to handle easily by researchers, there are great programs to help the researchers analyze such voluminous data without getting overloaded. The Cambridge Structural Database, which contains over 500,000 structures, has automated programs to search efficiently and its free Mercury program is great for analyzing individual molecules and their packing so effortlessly. The Inorganic Crystal Structure Database (ICSD), which contains over 130,000 structures, is specifically for inorganic structures. The Protein Data Bank (PDB)

It is also important to point out that there are a few drawbacks to this method, just like for any other method. The major problem is that one needs to get a good quality single crystal. The minor problems are that the hydrogens are not located accurately, and the structural details are accurate only to the solid state and may possibly deviate in solution or liquid state. As explained below, these are not of any serious concern.

As the technology and instrumentation advance, we are able to deal with smaller and smaller crystals, with only human handling ability making the limit. Since we bought the new instrument a decade ago, the recent advances make it possible to use just onetenth the size of the crystal we need in our 'old' instrument. Though synchrotron radiation sources can provide great data with even smaller crystals, those sources are

Though the hydrogens can be located more accurately using neutron diffraction, those facilities are fairly rare due to the need for nuclear radiation. However, the lost

The structure of the molecules may deviate to some extent in solution, but the crystal structure shows some of the energetically favored form for the molecules. The structure determined in the crystal will be present in solution, if not 100%, to a significant extent. We have had multiple isomers present in solution and only one isomer in solid, with the crystalline structure being the major isomer in solution, though in one case the solution isomer and crystal isomer were totally different. The intermolecular interactions will provide a statistically significant part of all the possibilities for non-crystalline states as

is the depository for protein structures with over 76,000 structures.

rare and costly, and not necessary for the most part.

accuracy is not at all a concern in the vast majority of situations.

When we come to this book on X-ray Crystallography, it is a compilation of recent advances in the structural studies in wide areas. The Chapters are divided into three Sections that deal with Small Molecules, Macromolecules, and Complimentary Methods.

Section 1 comprises structural studies of small molecules varying from organic molecules to metal complexes. This Section also includes polymorph studies on drug molecules and database analyses for weak interactions. Chapter 1 deals with the conformational analysis of acetyl anthracenes both by single crystal X-ray studies and theoretical studies. Chapter 2 discusses the structural studies of the less-studied calix[8]arenes with strategies to obtain single crystals in this challenging system. Chapter 3 explores the crystal engineering and co-crystallization aspects on polymorphs with emphasis on pharmacological relevance with examples. Chapters 4 and 5 examine the NH---X and Te---X (X=halogen) weak interactions by meta-analyses of Cambridge Structural Database.

Chapters 6-11 deal with structural studies of metal complexes. Chapter 6 focuses on the complexes of dioxolene (quinone based) ligands with various metals. Chapter 7 discusses the copper(I) complexes of Schiff base ligands whereas Chapter 8 analyses the structures of copper-halide binary dianions (CuX4 and Cu2X6). Chapters 9 and 10 examine the ruthenium complexes with the focus on pyridine based ligands and functionalized pyridylamine based ligands respectively. Chapter 11 discusses the structures of cyclometallated luminescent platinum complexes.

Section 2 covers the structural studies of proteins, including bioinformatics analysis of membrane proteins. Chapter 12 explores the effects of protein-noble gas interactions by structural analysis of data collected for crystals under various pressures of inert gases, which has implications on anesthesia and neuroprotection. Chapter 13 discusses the structural aspects of isocitrate dehydrogenase family of proteins. Chapter 14 explores the structures of autophagy (intracellular bulk degradation) related proteins. Chapter 15 shows the use of experimental/computational multidisciplinary approach based on structural studies, bioinformatics, modelling and model-guided biology experiments to obtain the structures and mechanisms of very difficult membrane proteins.

In Section 3, SAXS, Raman Microscopy, and NMR methods that compliment single crystal methods for difficult macromolecule systems are discussed. Chapter 16 describes a novel method to study macromolecule colloidal solutions by small angle scattering (SAXS) as a new way to get structural information in native state, especially in comparison to a known structure. Chapter 17 uses Raman microscopy to monitor the chemical modification of derivative proteins, either on the X-ray instrument or off the instrument (in-situ or ex-situ). Chapter 18 provides a novel NMR method (DIORITE based on TROSY) to study the morphological changes of proteins in solution by comparing with their solid state structures.

#### XIV Preface

I am sure that these Chapters will provide very a useful analysis/update on the various fields covered. Some of the Chapters will be useful for all researchers interested in structural analysis.

I thank my wife and long time coworker Natalya Timosheva for her assistance in all steps of this book. Also, I would like to thank InTech's Publishing Process Manager Martina Durovic for her immense help in the reviewing and editing processes.

With Best Wishes,

#### **Dr. Annamalai Chandrasekaran ("Chandra") Research Professor**

Massachusetts Center for Renewable Energy Science and Technology (MassCREST) Department of Chemistry University of Massachusetts Amherst USA
