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Learning Materials in Biosciences
Tim Skern
Exploring Protein
Structure:
Principles and
Practice
Learning Materials in Biosciences
Learning Materials in Biosciences textbooks compactly and concisely discuss a specific biological, bio-
medical, biochemical, bioengineering or cell biologic topic. The textbooks in this series are based on lec-
tures for upper-level undergraduates, master’s and graduate students, presented and written by
authoritative figures in the field at leading universities around the globe.
The titles are organized to guide the reader to a deeper understanding of the concepts covered.
Each textbook provides readers with fundamental insights into the subject and prepares them to indepen-
dently pursue further thinking and research on the topic. Colored figures, step-by-step protocols and take-
home messages offer an accessible approach to learning and understanding.
In addition to being designed to benefit students, Learning Materials textbooks represent a valuable tool
for lecturers and teachers, helping them to prepare their own respective coursework.
Exploring Protein
Structure: Principles
and Practice
Tim Skern
Max F. Perutz Laboratories
Medical University of Vienna
Vienna, Austria
This Springer imprint is published by the registered company Springer International Publishing AG,
part of Springer Nature
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
V
Acknowledgements
My course at the University of Vienna, and hence this book, was inspired by reading
“Introduction to Protein Structure” by Carl-Ivar Brändén and John Tooze.
I am grateful to the support of many students and colleagues. I would specially like to
thank Xué Strobl, Anna C. Schrempf and Brooke Morriswood, who patiently read all
the chapters and tested the PyMOL scripts. I would like to thank Martina Aumayr, Dan-
iel Azar, Gustavo Bezerra, Nina Bobik, Sofiya Fedosyuk, Karin M. Olek, Marina Pletzer
and Amelie Schoenenwald, members of my research group, who read through texts,
made valuable suggestions and let me use illustrations. Martin Groznica and Samar
Osmen, who took my course on the structure and function of proteins in 2017, took the
time to read the first chapter. Dieter Blaas, Mate Somlyay and Gang Dong, three col-
leagues from MFPL, read through texts on specific technical aspects. Florian Schur
from IST Austria significantly improved 7 Chap. 9. Thank you. Any errors in the book
are my responsibility.
I am indebted to Rachel Kramer Green and David Goodsell for permission to use screen-
shots from the RCSB PDB, to Roman Laskowski to use material from the PDBsum data-
base at EBI, to Sameer Velankar to use material from the EBI services and to Liisa Holm
to use the DALI server in Helsinki in the book. Christoph Gille allowed me to use the
STRAP progam and prepared a printable version of . Fig. 9.3. I also thank all the above
colleagues for their support in using their services. Rita Podzuna from Schrödinger, Inc.,
helped me with the copyright for PyMOL.
Marita Pollak provided terrific administrative support to keep chaos from the door and
conscientiously ordered all the figures, tables and boxes.
I thank Amrei Strehl from Springer Nature, who first suggested I write a book based on
my lecture course and who encouraged me to keep going even when I failed to adhere to
deadlines.
The work in my lab that is mentioned in this book was financed by the Austrian Science
Fund (FWF) and the Medical University of Vienna. The FWF also funded two doctoral
programs in structural biology for which some of the exercises in this book were
prepared.
VII
Contents
3 Exploring Fundamentals............................................................................................................ 29
3.1 Background............................................................................................................................................. 30
3.2 The Structure of Hemoglobin.......................................................................................................... 32
3.3 The Plant Protease Papain................................................................................................................ 36
3.4 Electrostatic Interactions................................................................................................................... 42
3.5 Hydrogen Bond Interactions Between Side-Chains................................................................ 47
3.6 Hydrogen Bonds in Tyrosyl-tRNA Synthetase........................................................................... 50
3.7 Van der Waals Interactions............................................................................................................... 52
3.8 Summary of PDB Information Discussed in This Chapter..................................................... 55
3.9 Summary of PyMOL Commands Introduced in This Chapter.............................................. 55
3.9.1 General and Settings............................................................................................................................ 55
3.9.2 Organization............................................................................................................................................ 55
3.9.3 Viewing..................................................................................................................................................... 55
3.9.4 Selecting................................................................................................................................................... 56
3.9.5 Coloring.................................................................................................................................................... 56
3.10 Further Reading.................................................................................................................................... 56
3.10.1 Books......................................................................................................................................................... 56
3.10.2 Online Resources................................................................................................................................... 56
3.11 Exercises................................................................................................................................................... 57
References............................................................................................................................................... 58
VIII Contents
Supplementary Information
Index........................................................................................................................................................... 251
1 1
This book developed from a course on protein structure and function that I
1 have been offering at the University of Vienna since 1999. The course arose
because I sensed a need to support my students to move forward from the
theoretical knowledge of protein structure obtained in their biochemistry
courses to a more active interaction that would enable them to make use of
the huge amount of information on macromolecular structures stored in the
Protein Data Bank (PDB). Students are used to seeing ribbon diagrams of
proteins and other macromolecules in publications. However, they usually are
unsure of how to interpret them and how they can be generated.
Most of the structures deposited in the PDB were solved by X-ray crystallography. The
foundations for this method of structure determination were laid just over 100 years ago
by Lawrence Bragg while, amazingly, still an undergraduate at Cambridge (Cruse 2014;
Hall 2014). However, it was not until 1958 that the first structure of a protein was deter-
mined (Kendrew et al. 1958). Progress was slow, so that even 10 years later, the solution
of the structure of the enzyme RNase A was hailed by the journal Nature as “an event of
unique importance and interest” (Anon 1967a). It is also enlightening to examine the
questions being posed on the nature of protein folds as the first structures became avail-
able (Anon 1967b). For instance, it was not clear at this time that the hydrophilic residues
would be on the exterior and the hydrophobic ones in the interior. Since the 1970s, the
number of macromolecular structures solved has been growing exponentially for many
years now. Now, at the time of writing (July 2017), the coordinates of over 130,000 mac-
romolecules have been deposited and made available to the community in the PDB, with
around 10,000 new structures being added per year. The growth in the PDB has been
mirrored by increases in the number of tools (EMBL-EBI 2017) and databases (Laskowski
2016) on the Internet to analyze the information in the PDB. At present, there are so
many algorithms performing so many different types of analyses that the situation is quite
bewildering for students starting out on their own.
My lecture series and hence this book set out to support bachelor, masters, and PhD
students in using the currently available software to analyze and utilize the information
in the PDB. The introduction to the PDB will not be exhaustive but should just reflect
the parts that a practicing scientists needs. The introduction should allow students to
interpret protein structures for their own seminars and research projects. The archived
information in the flat files of the PDB should come alive and provide the starting point
for biological investigations. There is so much to discover about macromolecular structure
that the students of today and tomorrow can rest assured that there will always be cutting-
edge research to do.
The course and this book are designed for students of all the life sciences, not just those
focusing directly on structural biology. The interpretation of macromolecular structures
can represent the starting point for research in fields as diverse as pharmacology, chem-
istry, genetics, biotechnology, virology, cell biology, parasitology, evolution, bioinformat-
ics, cancer, and medicine. The research can range from the design of drugs to combat
human immunodeficiency virus (HIV) infections and thus fight AIDS to the modulation
of enzymatic specificity (Wells and Estell 1988). Many of the drugs used to treat AIDS
today were designed and improved using the structure of the virally encoded protease that
HIV uses to process its maturing viral particles. . Figure 1.1 shows the overall structure
of the HIV-1 protease bound to an inhibitor. The protease is a homodimer (i.e., made up
of two identical chains) with the inhibitor lying between the monomers. Have a close look
Chapter 1 · The Rationale Behind This Workbook
3 1
NB
CA
CB
NA
.. Fig. 1.1 Cartoon drawing of the HIV-1 protease bound to a substrate-based inhibitor using the PDB
entry 4hvp and the software PyMOL. The two chains of the homodimer are colored in the spectrum of
the rainbow; the N- and C-termini of the two chains (A and B) are labeled. The inhibitor is shown as sticks
and is colored according to atoms: carbon is in yellow, oxygen in red, and nitrogen in blue. Note that
PyMOL draws the β-sheets as long arrows indicating the direction of the polypeptide chain. The com-
mands to draw this image can be found in 7 Chap. 2, 7 Box 2.3. All diagrams in this book were labeled
with a standard graphics software unless otherwise stated
.. Table 1.1 PDB entries illustrated in this chapter. Every entry has its own four character
identifier
at the protein-inhibitor complex. Is there anything unusual about the symmetry? We will
look at this structure more closely in 7 Sect. 2.1 using the PDB entry 4hvp mentioned in
. Table 1.1.
Further support for the importance of understanding macromolecular structure comes
from the work of neurophysiologist and biophysicist Roderick MacKinnon. MacKinnon is
a superb example of a scientist who demonstrates that structural biology need not always
be carried out by scientists who have in-depth training in this area. MacKinnon began to
use structural biology in the 1990s to further his studies of proteins that allow the move-
ment of potassium ions across membranes. During his time as a researcher at Harvard
University, he started to learn how to perform X-ray crystallography on proteins; subse-
quently, he set up his own X-ray crystallography laboratory at the Rockefeller University in
New York City and eventually succeeded in solving the structure of a bacterial potassium
4 Chapter 1 · The Rationale Behind This Workbook
1 a b
N N
N N
C C
C C
.. Fig. 1.2 Cartoon images of the potassium channel from Streptomyces lividans drawn using the PDB
entry 1bl8 and the software PyMOL. a The polypeptide of each monomer of the tetramer is in a differ-
ent color. Positions of the N- and C-termini are indicated. b Each monomer is colored as a rainbow from
N- (blue) to C- (red) terminus. In both images, the three potassium ions are in gray. Image A shows the
arrangement of the chains to each other; image B shows the direction of polypeptide chain from the
N- to the C-terminus. The commands to draw these images are in 7 Box 2.4
channel in 1998 (. Fig. 1.2). MacKinnon was awarded the Nobel Prize for this work in
2003 (MacKinnon 2004). His laboratory has solved structures of many more transporter
proteins in the last 15 years.
A similar advance for the field of pharmacology was the determination in 2011 by
Brian Kobilka and his group of several structures of membrane proteins that recognize
effector molecules such as adrenaline and opioids (e.g., morphine) (Rasmussen et al. 2011;
Manglik et al. 2012). The work on the adrenaline receptors (termed adrenergic receptors)
won Kobilka the Nobel Prize in 2012 (Kobilka 2013). Kobilka’s work is also noteworthy
for the number of techniques that he used to determine the structures. The techniques
were summarized in a Nature “News and Views” article that accompanied the publication
of his work and enabled the nonspecialist to grasp the advance in knowledge (Schwartz
and Sakmar 2011). This Nature commentary, along with a contemporary one from the
journal Science (Ward et al. 2013), shows not only that the amount of data in the PDB and
the software to analyze this data are increasing but also that there is a third area of rapid
growth, namely in the number of techniques that need to be mastered for structural biol-
ogy itself. Indeed, the authors suggest that the paper describing the adrenergic receptor
should be the basis of a semester-long course in graduate school on how these techniques
are integrated in structural biology. In this vein, 7 Chap. 9 of this book enables you to
References
5 1
Take Home Message
In summary, the aim of this book is to introduce appropriate tools to enable you to
understand and make sense of the information in the PDB as well as providing infor-
mation on the integration of the techniques used today (Ward et al. 2013). Enormous
and exciting advances are being made in structural biology at present (Berndt and
Deisseroth 2015; Kang et al. 2015; Hemmer and Gomes 2015; Callaway 2015), includ-
ing the determination at the near-atomic level of structures of the ribosome (Ramak-
rishnan 2014), the spliceosome (Cate 2016), the nuclear pore complex (Lin et al. 2016),
and the injectisome complex of the pathogenic bacteria Salmonella typhimurium
(Worrall et al. 2016). Many of these structures were solved using recent advances
in cryo-electron microscopy (cryo-EM) (Callaway 2015). However, it is not just large
supramolecular complexes that can be solved by this technique. Smaller proteins
such as β-galactosidase (molecular mass 160 kDa) and hemoglobin (molecular mass
64 kDa) have been solved using cryo-EM at resolutions that allow the backbone and
side chains to be positioned (Bartesaghi et al. 2015; Khoshouei et al. 2017).
explore some recently determined structures, including Kobilka’s structures of the adren-
ergic receptor, and to become acquainted with some of the new techniques now becoming
established in structural biology.
This book is intended to encourage you to analyze the macromolecular structures yourself
and wonder about the biological secrets that they reveal.
References
Anon (1967a) Ribonuclease structure – some implications. Nature 213:960
Anon (1967b) Structure and function of proteins. Nature 215(5105):1066–1067
Bartesaghi A, Merk A, Banerjee S, Matthies D, Wu X, Milne JL, Subramaniam S (2015) 2.2 A resolu-
tion cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor. Science
348(6239):1147–1151. https://doi.org/10.1126/science.aab1576
Berndt A, Deisseroth K (2015) Optogenetics. Expanding the optogenetics toolkit. Science 349(6248):
590–591. https://doi.org/10.1126/science.aac7889
Callaway E (2015) The revolution will not be crystallized: a new method sweeps through structural biol-
ogy. Nature 525(7568):172–174. https://doi.org/10.1038/525172a
Cate JH (2016) Structure. A big bang in spliceosome structural biology. Science 351(6280):1390–1392.
https://doi.org/10.1126/science.aaf4465
Cruse M (2014) 100 years of crystallography. Biochemist 36(1):40–42
Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R (1998)
The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science
280(5360):69–77
Hall KT (2014) The man in the monkeynut coat: William Astbury and the Forgotten Road to the Double-
Helix. Oxford University Press, Oxford, U.K.
Hemmer P, Gomes C (2015) Physics. Single proteins under a diamond spotlight. Science 347(6226):1072–
1073. https://doi.org/10.1126/science.aaa7440
Kang Y, Zhou XE, Gao X, He Y, Liu W, Ishchenko A, Barty A, White TA, Yefanov O, Han GW, Xu Q, de Waal
PW, Ke J, Tan MH, Zhang C, Moeller A, West GM, Pascal BD, Van Eps N, Caro LN, Vishnivetskiy SA,
Lee RJ, Suino-Powell KM, Gu X, Pal K, Ma J, Zhi X, Boutet S, Williams GJ, Messerschmidt M, Gati C,
Zatsepin NA, Wang D, James D, Basu S, Roy-Chowdhury S, Conrad CE, Coe J, Liu H, Lisova S, Kupitz C,
Grotjohann I, Fromme R, Jiang Y, Tan M, Yang H, Li J, Wang M, Zheng Z, Li D, Howe N, Zhao Y, Standfuss
6 Chapter 1 · The Rationale Behind This Workbook
J, Diederichs K, Dong Y, Potter CS, Carragher B, Caffrey M, Jiang H, Chapman HN, Spence JC, Fromme P,
1 Weierstall U, Ernst OP, Katritch V, Gurevich VV, Griffin PR, Hubbell WL, Stevens RC, Cherezov V, Melcher
K, Xu HE (2015) Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature
523(7562):561–567. https://doi.org/10.1038/nature14656
Kendrew JC, Bodo G, Dintzis HM, Parrish RG, Wyckoff H, Phillips DC (1958) A three-dimensional model of
the myoglobin molecule obtained by X-ray analysis. Nature 181(4610):662–666
Khoshouei M, Radjainia M, Baumeister W, Danev R (2017) Cryo-EM structure of haemoglobin at 3.2 A
determined with the Volta phase plate. Nat Commun 8:16099. https://doi.org/10.1038/ncomms16099
Kobilka B (2013) The structural basis of G-protein-coupled receptor signaling (Nobel Lecture). Angew
Chem Int Ed Engl 52(25):6380–6388. https://doi.org/10.1002/anie.201302116
Laskowski RA (2016) Protein structure databases. Methods Mol Biol 1415:31–53. https://doi.
org/10.1007/978-1-4939-3572-7_2
Lin DH, Stuwe T, Schilbach S, Rundlet EJ, Perriches T, Mobbs G, Fan Y, Thierbach K, Huber FM, Collins
LN, Davenport AM, Jeon YE, Hoelz A (2016) Architecture of the symmetric core of the nuclear pore.
Science 352(6283):aaf1015. https://doi.org/10.1126/science.aaf1015
MacKinnon R (2004) Potassium channels and the atomic basis of selective ion conduction (Nobel Lecture).
Angew Chem Int Ed Engl 43(33):4265–4277. https://doi.org/10.1002/anie.200400662
Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK, Pardo L, Weis WI, Kobilka BK,
Granier S (2012) Crystal structure of the micro-opioid receptor bound to a morphinan antagonist.
Nature 485(7398):321–326. https://doi.org/10.1038/nature10954
Miller M, Schneider J, Sathyanarayana BK, Toth MV, Marshall GR, Clawson L, Selk L, Kent SB, Wlodawer
A (1989) Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A
resolution. Science 246(4934):1149–1152
Ramakrishnan V (2014) The ribosome emerges from a black box. Cell 159(5):979–984. https://doi.
org/10.1016/j.cell.2014.10.052
Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D,
Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara
RK, Kobilka BK (2011) Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature
477(7366):549–555. https://doi.org/10.1038/nature10361
Schwartz TW, Sakmar TP (2011) Structural biology: snapshot of a signalling complex. Nature 477(7366):
540–541. https://doi.org/10.1038/477540a
Tools & Databases (2017) EMBL-EBI. http://www.ebi.ac.uk/services/all. Accessed 15 Feb 2017
Ward AB, Sali A, Wilson IA (2013) Biochemistry. Integrative structural biology. Science 339(6122):913–915.
https://doi.org/10.1126/science.1228565
Wells JA, Estell DA (1988) Subtilisin – an enzyme designed to be engineered. Trends Biochem Sci
13(8):291–297
Worrall LJ, Hong C, Vuckovic M, Deng W, Bergeron JR, Majewski DD, Huang RK, Spreter T, Finlay BB, Yu
Z, Strynadka NC (2016) Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome
basal body. Nature 540:597–601. https://doi.org/10.1038/nature20576
7 2
2.6 Exercises – 25
References – 27
We are now living in an atomic age. In order to understand the world, every person needs
to have some understanding of atoms and molecules. Linus Pauling and Roger Hayward
(Pauling and Hayward 1964)
2 What You Will Learn from This Chapter
This chapter will first introduce you to the freely accessible data bank of protein structures
(the “PDB”) and show you how to search, access, and understand the information within it.
The second half presents a tool, PyMOL, with which you can start to visualize and analyze
the data stored in the PDB (. Table. 2.1).
To provide a basis for the exploration of protein structures, let us start by looking at the
origins of the Protein Data Bank (PDB). The PDB was initiated in the late 1960s and early
1970s by a group of pioneering scientists who were solving the first structures of proteins
by X-ray crystallography. The group desired to make the coordinates of the structures they
had determined available to the research community. Their solution was to found the PDB
as reported in Nature (Anon 1971). By 1976, 13 structures had been deposited in the PDB
and released to the community via magnetic tape. More information on the origins and
aims of the PDB is provided in an article written by Helen Berman, one of the founders,
and colleagues on the occasion of the 40th anniversary of the PDB in 2011 (Berman et al.
2012). Selected presentations of scientists involved in setting up the PDB can be found at
7 https://www.wwpdb.org/about/outreach-content/pdb40.
Since its inception, the PDB has grown enormously. As mentioned in the previous
chapter, the number of entries now totals over 130,000. . Figure 2.1 illustrates the growth
of the number of entries in the PDB since the 1970s. A marvelous drawing of 200 differ-
ent icosahedral viruses from their PDB entries produced for the 200th “Molecule of the
Month” feature of the portal RCSB illustrates how extensive the PDB has become (7 http://
pdb101.rcsb.org/learn/resource/200-icosahedral-viruses-poster) (Goodsell et al. 2015).
About 90% of the structures in the PDB have been determined using X-ray crystallogra-
phy, 9% by nuclear magnetic resonance (NMR), and 1% by electron microscopy (EM) (see
7 http://www.rcsb.org/pdb/statistics/holdings.do). However, the numbers of structures solved
by NMR and especially EM are rising rapidly. This development can be clearly seen when the
entries are grouped according to the experimental method used in the “Content Growth” sec-
tion at 7 http://www.rcsb.org/pdb/static.do?p=general_information/pdb_statistics/index.html.
140000
120000
100000
80000
Entries per year
60000 Total entries
40000
20000
0
1976 1981 1986 1991 1996 2001 2006 2011 2016
.. Fig. 2.1 Growth of total entries in the PDB. (Data taken from the PDB (7 http://www.rcsb.org/pdb/
statistics/contentGrowthChart.do?content=total&seqid=100))
7 Section 9.3 of 7 Chap. 9 look at a recent structure of the spliceosome that was solved by EM
(7 Sect. 9.3).
How many of the 130,000 entries in the PDB are unique? That is not an easy question
to answer. Many structures just differ in one amino acid or in the presence or absence of a
particular ligand. In addition, two entries may contain the same protein, but the structures
were determined from two different crystal forms. To provide an answer to the number
of unique structures, the curators of the PDB have compared the protein sequences of
protein structures deposited in the PDB at various levels of sequence identity (7 http://
www.rcsb.org/pdb/statistics/clusterStatistics.do); the lower the level of sequence identity,
the fewer the number of unique sequences that will be defined (i.e., protein sequences
showing 70% identity or more will be counted as the same entry). With an identity of
90%, one finds approximately 40,000 unique sequences and thus unique structures in the
PDB (Holm and Laakso 2016). With an identity of 70%, the number falls to about 25,000,
fivefold fewer than the total number of entries. Nevertheless, 25,000 unique structures still
represent a huge amount of information.
The information stored in the PDB is available online at four different entry portals
(listed in . Table 2.2).
10 Chapter 2 · An Archive and a Tool: PDB and PyMOL
.. Fig. 2.2 The entry page of the RCSB (7 www.rcsb.org) site to the PDB downloaded on December
7, 2017 (Berman et al. 2000; Rose et al. 2017). All Internet sites mentioned in this book were successfully
used with Firefox and with Google Chrome on Windows and Mac platforms
The information archived at the four sites is the same. However, the presentation of the
information on the entry pages and their user-friendliness differ quite considerably. I find
the site at the Research Collaboratory for Structural Bioinformatics (RCSB; 7 www.rcsb.
org) the most useful. Consequently, all exercises involving the PDB in this book start from
this link. The entry page of the RCSB is shown in . Fig. 2.2.
I prefer the 7 www.rcsb.org entry page for the following four reasons. First, there is
a direct window at the top for searching the database (. Fig. 2.2). Second, there is the
2.1 · The Protein Data Bank (PDB)
11 2
.. Fig. 2.3 The top of the “Structure Summary” for the PDB entry 4hvp downloaded on December 7,
2017 (Berman et al. 2000). The red arrow points to the buttons for the displaying and downloading of the
PDB files and the green one to the buttons to access the reports mentioned in the text
molecule of the month feature mentioned earlier which includes background and draw-
ings of selected macromolecules and is terrific for new students to browse through. Third,
there is a direct link to a feature called “PDB-101” which is another site where new stu-
dents might start to find a basic introduction to the PDB. The fourth reason is that the
search results are presented clearly with just the amount of information required to decide
whether the entry is the one being sought. We can see this by searching for the PDB file that
was used to make the image of HIV-1 protease (. Fig. 1.1) shown in the previous chapter
on the rationale behind this book. Type the PDB identifier “4hvp” into the search window
followed by “enter.” The structure summary for the entry 4hvp appears (. Fig. 2.3). The
top of the structure summary page provides an image of the structure, information on
the authors, the date of deposition, and the publication (with a direct link to PubMed)
describing the structure as well as the quality and validity of the structure determination.
The validation and quality reports of the structures archived in the PDB are a high
priority of the curators. Without these reports, the users of data cannot rely on the accu-
racy of the entries, and the database becomes useless. We can see links (indicated with a
green arrow) to two representations of the data on the summary page, namely, the recently
12 Chapter 2 · An Archive and a Tool: PDB and PyMOL
introduced 3D report and the more detailed full report. We will look at these as we go
through the book (e.g., 7 Sects. 3.2 and 6.2).
Scroll down the 4hvp structure summary for more information on both the protein
2 (the macromolecule) and ligands such as the inhibitor (small molecules) as well as links
to other databases. There is already an enormous wealth of information on this summary
page. Above the image and the PDB identifier, the tabs provide more detailed information
than that on the summary page or, in some cases, group the same information in a differ-
ent way. Indeed, this single summary page with its associated tabs gives us an insight into
how much information is contained in the PDB (remember that this page is just one of
over 130,000 entries). From this one entry, we can appreciate two of the greatest challenges
for both teachers and students in the age of big data: how do teachers teach this material
and how do students understand, analyze, and profit from it in their class assignments and
research projects?
So, we have the PDB entry for 4hvp. What shall we do with it? Let us start with the file
containing the coordinates that were used to make the image in . Fig. 1.1. After all, the
PDB started as a repository of the atomic coordinates, so they should be somewhere in the
file. To find them, click on “Display File” (marked with a red arrow in . Fig. 2.3) and then
“PDB format.” . Figure 2.4 shows the top of the file.
The top of the PDB format file provides background information on the structure.
Scroll down the file and you will see a large quantity of information on the structure deter-
mination, the quality of the structure, the angles of the bonds, the residues involved in sec-
ondary structure, and some symmetry operations. The word at the beginning of each line
indicates the content in that line (or record, as it is known). We will look at some of the
contents of the records as we go through the book. For now though, we wish to look at the
coordinates which are toward the bottom of the file. In . Fig. 1.1, we observed two protein
chains and one molecule of the inhibitor. We should therefore expect coordinates for two
polypeptide chains and for one molecule of inhibitor. To find these coordinates, scroll
down the 4hvp file until you see records prefixed with “ATOM” (see . Fig. 2.5). These
contain the x, y, and z coordinates of the carbon, oxygen, nitrogen, and sulfur atoms of
the macromolecular structure. . Figure 2.5 shows the fields for the first two amino acids
and explains the meaning of all the entries in each column. To localize the coordinates of
chains A and B and their ligands, we need to focus on column E. This column shows the
letter A for the first two amino acids, indicating residues of chain A, the first of the chains.
The lines for the second chain, chain B, can be found by scrolling further down. At the
very end of the file, there are lines prefixed with HETATM (heterologous atom); these
contain the x, y, and z coordinates of the atoms of small molecules (these are often ligands
specifically bound to the protein) that could be observed in the electron density but which
are not part of the protein or nucleic acid. In the entry 4hvp, these are the molecule of the
inhibitor (termed 2NC), which has been designated as amino acid 0 of chain B, as well as
a substantial number of water molecules.
When I was examining this PDB file for this book, I was surprised to notice HETATM
lines among those for the coordinates of the protein itself. Look at residues 67 and 95 in
both chains. The amino acid is given as ABA, short for aminobutyric acid. This amino
acid, which is not normally found in proteins, is present here because the protein (which
2.1 · The Protein Data Bank (PDB)
13 2
.. Fig. 2.4 The top of the PDB file for 4hvp. (Berman et al. 2000)
14 Chapter 2 · An Archive and a Tool: PDB and PyMOL
A B C D E F G H I J K L
ATOM 1 N PRO A 1 -3.358 7.992 34.537 1.00 15.57 N
ATOM 2 CA PRO A 1 -2.420 7.030 35.105 1.00 15.88 C
2 ATOM
ATOM
3
4
C
O
PRO
PRO
A
A
1
1
-1.038
-0.920
7.006
7.437
34.472
33.309
1.00
1.00
14.49
14.48
C
O
ATOM 5 CB PRO A 1 -3.076 5.683 34.785 1.00 15.99 C
ATOM 6 CG PRO A 1 -3.651 5.927 33.414 1.00 16.69 C
ATOM 7 CD PRO A 1 -4.063 7.389 33.400 1.00 16.19 C
ATOM 8 N GLN A 2 -0.130 6.424 35.234 1.00 13.19 N
ATOM 9 CA GLN A 2 1.249 6.329 34.731 1.00 13.76 C
ATOM 10 C GLN A 2 1.534 4.886 34.368 1.00 12.96 C
ATOM 11 O GLN A 2 1.391 4.030 35.237 1.00 13.56 O
ATOM 12 CB GLN A 2 2.241 6.885 35.716 1.00 13.72 C
ATOM 13 CG GLN A 2 3.728 6.627 35.543 1.00 15.82 C
ATOM 14 CD GLN A 2 4.582 7.612 36.299 1.00 17.54 C
ATOM 15 OE1 GLN A 2 4.804 7.575 37.492 1.00 21.95 O
ATOM 16 NE2 GLN A 2 5.143 8.603 35.625 1.00 20.65 N
.. Fig. 2.5 The atomic coordinates of the first two amino acids of the HIV-1 protease in entry 4hvp.
The contents of the columns are as follows: A Entity (ATOM, TER (=end of chain), HETATM). B Atom num-
ber. C Atom name. N, CA, C, and O are the backbone atoms, and the remainder are side-chain atoms.
Thus, CB, CG, and CD are the names of the carbon atoms of the glutamine side-chain, OE1 and NE2 of
the amide group. D Residue name. E Chain identifier (scroll down the PDB file to see chain B). F Residue
number. G, H, I x, y, and z coordinates, respectively. J Occupancy of the atom. Here, the number in every
record is 1.00, indicating that each atom is always in the same position. Sometimes, in a crystal structure,
the side-chain of an amino acid can adopt two conformations. This number in column J, as we will see
in 7 Sect. 4.4.2, reflects how many of the atoms are in each of the two conformations. K The B factor or
the temperature factor or displacement factor describes the motion of the atom in the crystal, as we
will see in 7 Sect. 7.4.2. Values below 10 mean that the atom is hardly moving; numbers of 40 or higher
indicate that the atom is very mobile. Surface atoms in proteins often have higher values. L Element
symbol
comprises just 99 amino acids) was small enough to be chemically synthesized. During
the synthesis, the two cysteines in each chain were replaced with ABA, as its aliphatic
side-chain is of the same length and size as that of the cysteine side-chain (Wlodawer et al.
1989). The scientists took this measure to avoid problems of oxidation and non-specific
interactions of cysteine residues.
You may be asking yourself why there are no coordinates for hydrogen atoms. The
answer lies in the nature of the technique used to generate the data for the protein struc-
ture. In X-ray crystallography, the electron density maps are produced by electron dif-
fraction of X-rays. As the hydrogen atom has only one electron, the chances of scattering
from it are almost negligible. Therefore, the positions of the hydrogen atoms cannot be
determined. You may, however, find some PDB files containing coordinates for hydrogen
atoms. These are sometimes added during the refinement of the structure. The structure
of RNase A with the PDB identifier 7rsa, which we will examine in 7 Chap. 4 (7 Sect. 4.3),
is an example.
We have found the coordinates of the entry 4hvp. This is a step forward, but we cannot
visualize and investigate the information stored without suitable software. The next section
introduces the program PyMOL (The PyMOL Molecular Graphics System, Schrödinger,
LLC).
2.2 · PyMOL
15 2
2.2 PyMOL
I have chosen PyMOL as the modeling software for this book for several reasons. First,
the program is available for free upon registration as an “educational-use-only” version
for students. The current “educational-use-only” version 1.74 (as of August 2015, used in
the making of this book) of PyMOL does not have every feature of the full version (e.g.,
it does not allow you to make publication-quality images because the “ray” command is
inactivated); nevertheless, it will enable you to produce all of the images in this book on
either a PC or a Mac. All of the drawings could be made by the two students who read
this book using the “educational-use-only” version. Of course, the high-resolution images
in this book were produced using a fully licensed version of PyMOL (version 1.8.4.2) on
Windows 10 but still using the same instructions for drawing them. Second, PyMOL can
fetch PDB files directly without having to download and save them. Third, PyMOL can
also read PDB files that reveal the quaternary structure of a protein. Fourth and most
importantly, the program is very powerful for drawing images, allowing the production of
pictures which have adorned the covers of many top journals. You can see some of these
images at the link 7 http://pymolwiki.org/index.php/Covers.
During the writing of this book, PyMOL 2.0 was released for which there is also an
“educational-use-only” version. However, as almost all information online is with the
older version and I have as yet no experience with the new release, the book still uses the
educational version 1.7.4. This earlier version can of course still be downloaded.
Nothing is however perfect, and PyMOL is no exception. If you scan the literature or
look at the record “REMARK 3” in the PDB, you will find that scientists use other pro-
grams such as SHELX (Sheldrick 2008), Coot (Emsley et al. 2010), and PHENIX (Adams
et al. 2010) to build proteins from electron density maps. If your project is going to involve
such experiments, then you may want to have a look at these programs. However, they are
outside the scope of this book, and you will thus be on your own.
Powerful software often means that it can take time for new users to be able to under-
stand how the program works and what they can do with it. If you follow the instructions
in the boxes and the videos of this book however, you should be able very rapidly to use the
software on your own. All of the instructions have been tested and improved by bachelor
or masters students with no prior experience of PyMOL. Indeed, using the approaches for
PyMOL described, such students working in my lab have within 2 to 3 days been able to
compare and superimpose the models of interactions proposed between two proteins by
the online protein docking algorithm ClusPro 7 https://cluspro.bu.edu/. The work of one
bachelor student using ClusPro and PyMOL provided the basis for a scientific publication
from my own group (Aumayr et al. 2017).
To install PyMOL with an “educational-use-only” license, you need to first register at
7 http://pymol.org/edu/?q=educational/ by filling out the form. As a student, you will be
asked for a supervisor, the degree you are studying for, and when you expect to complete
it; you can enter “self-study” if you cannot give a name of a supervisor. Once you have
completed the form, you will receive an email with a username and password. Download
the appropriate program for your operating system and install the program, following the
prompts. You do not need to change the default settings to add extra files that PyMOL
can accept. In the list of programs on a PC, you may have different options to start the
program. For the exercises in this workbook, always use “PyMOL” to start.
16 Chapter 2 · An Archive and a Tool: PDB and PyMOL
With PyMOL now installed, you can begin to make the image of the HIV-1 protease
shown in . Fig. 1.1. First, we need to download the PDB file with the coordinates. Click
on the “Download File” option in the summary page of the 4hvp entry (see . Fig. 2.3), and
2 choose “PDB format.” If you are offered the option to always open PDB files with PyMOL,
then do so. If not, select the “Save” option, and save the file as you wish. Once saved, click
on the PDB file, and take PyMOL as the program to always open such files. PyMOL will
open and give you your first look at the structure of the entry 4hvp, most likely in a mass of
colored sticks that looks very uninformative as shown in . Fig. 2.6a (Mac) and . Fig. 2.6b
(Windows). 7 Boxes 2.1 and 2.2 introduce you to some PyMOL basics.
.. Fig. 2.6 The PyMOL program after opening the PDB entry 4hvp using Mac a and Windows 10 b
operating systems. On the Mac version, the two frames are linked together, whereas on Windows, they
are separate. Note the drop-down menu at the top, the two command lines with the PyMOL prompts,
and the graphical user interfaces on the top and bottom right in the lower frames. 7 Boxes 2.1 and 2.2
explain some important features. Please familiarize yourself with them before you start
2.2 · PyMOL
17 2
b
4. The GUI at the bottom right has many features. We will need in this book the “S” button
which shows the sequence of the molecule above the structure and the “F” botton to go full
screen. The “selecting” tool lets us run through the options you can select (residues, chains,
2 segments, objects, molecules, C-alphas, atoms, and then back to residues). Clicking on “view-
ing” or “buttons and keys” brings up “editing.” We will rarely need “editing” in this book; if
something does not work, check that you are not on editing by mistake.
5. The lower GUI also describes how the mouse buttons can be used to manipulate the struc-
ture drawn by PyMOL. However, as this looks rather complex at first, here are the basic set-
tings for the mouse on a MacBook with an external monitor and a laptop running Windows
10 with an external monitor.
55 Rotate: left mouse button
55 Zoom: right mouse button
55 Move across screen: press down on mouse wheel (on a Mac, plus ⌘)
55 Adjust slab: mouse wheel
55 The slab command adjusts the thickness of the view through the molecule, thus let-
ting you see more or less of it. If any of these settings do not work on your setup, try
some combinations of the above keys yourself.
6. You can also use your device’s touchpad. However, this requires some practice, and I suggest
you gain some experience with a mouse first.
Box 2.2 Using the Mouse in PyMOL to Select Atoms, Residues, Chains, and Molecules
Items selected in PyMOL are indicated by small pink squares; an item “(sele)” also appears in the
GUI at the top right. The mouse in PyMOL has many features for selecting atoms, residues, chains,
and molecules. Here are some important ones. First though, use the mouse to click on the S but-
ton on the lower right GUI in your image of 4vhp.
1. Using the mouse on the amino acid sequence at the top
1.1 Set the GUI selecting tool on the bottom right to “residues.” Clicking on an amino acid
with:
The left button shows the positions of the atoms from that amino acid.
The middle button centers the molecule on that amino acid.
The right button pops up a menu which is a subset of the GUI commands at the top right.
The mouse can also be dragged to select any number of residues in the sequence at the
top. The above commands then work for all amino acids in that selection.
1.2 Set the GUI selecting tool on the bottom right to “chains.” The above commands work as
for “residues” except that only chains can be selected.
2. Using the mouse on the molecule itself. Again it depends on whether atom, residue, or chain
is selected in the bottom right-hand GUI. The commands here are given for residues.
A single click with the left button shows the positions of the atoms from that amino acid
and highlights the residue in the sequence at the top. A second click with the left button
deselects. A left click immediately followed by a right click brings up a menu allowing you
to perform operations on the selected amino acids. A rapid double-click with the left button
identifies the clicked atom and brings up an activity menu.
Clicking with the middle button centers the molecule on that amino acid.
A single click with the right button has the same effect as a rapid double-click with the
left button.
As mentioned for structures in 7 Box 2.1, selections can also be disabled and enabled by
clicking on the bar on the top right GUI called “sele”. In addition, a selection can be disabled
by clicking on the background. Be careful, though; even though you cannot see the selec-
tion, the items are still selected.
3. Using the mouse on the background of the drawing
Double-clicking with the left mouse or single-clicking with the right mouse brings up
the main pop-up menu that allows several basic operations to be performed.
2.2 · PyMOL
19 2
You can achieve a great deal in PyMOL just by using the GUI and the mouse. However,
I suggest you learn to use the command line because it is more rapid, more versatile, and
more accurate in selecting specific parts of macromolecules. Using the command line also
helps you to understand what the algorithms are doing and should give you a deeper
understanding of the structure of the macromolecules. New commands are introduced
with comments in each chapter, and the newly introduced commands are summarized at
the end of each chapter. There is also support from PyMOL itself by using certain com-
mands and keys. If you need help on a command (e.g., fetch), type in “help fetch” and
“return” to find information on the command. If you are not sure how a command is
called, type, for instance, “help fe,” and hit the “tab” button; PyMOL will give all possible
commands starting with “fe”. This “tab completion” also works if you just want to run the
command itself but are not sure of the name. You can also find the syntax for a command
(e.g., show) by typing “show?”. The reports given by PyMOL to these suggestions may
sound complex now, but if you follow the suggestions in this book, they will soon become
familiar.
Now, let us use both the command line and the mouse to turn the rather daunting
presentations in . Fig. 2.6 into the image in . Fig. 1.1. To do this, follow carefully the
instructions in 7 Box 2.3. Are you surprised that you needed so few commands to produce
the image? In the penultimate step, we changed the background color from black to white.
If you are showing an image in a seminar or tutorial, a black background is generally bet-
ter, whereas white is preferred for printed images.
In the boxes in this book, information and explanation is preceded by the hash tag
(#). Commands for an Internet browser, the mouse, the PyMOL command line, and the
PyMOL GUI (graphical user interface) are clearly indicated. All commands in the com-
mand line require a return (“enter”) to take effect. Clicks with the left mouse button are
marked with “>”.
Box 2.3 Drawing the HIV-1 Protease in . Fig. 1.1 starting from . Fig. 2.6
# entry 4hvp is already loaded in Fig. 2.6
# show as cartoon. All commands require enter to take effect
PyMOL> as cartoon
# show the sequence of the entry
GUI> Click on the “S” button in the bottom right corner
# color the cartoon of each chain as a rainbow spectrum from blue at the N-termi-
nus to red at the C-terminus (using the utility command “rainbow”). “Residue” in
PyMOL actually stems in PyMOL from the expression “residue-identifier-list.”
PyMOL> util.rainbow chain A and residue 1–99
PyMOL> util.rainbow chain B and residue 1–99
# show the inhibitor as sticks. It is designated residue 0. With the slider under the
amino acid sequence, look for 2NC at the end of chain B
PyMOL> show sticks, residue 0
# color the inhibitor with C yellow; O, red; and N, blue using the utility command
“cbay”: color by atom, carbon yellow. “Residue” can be further shortened to “resi”
or “i.” for “identifier”.
PyMOL> util.cbay i. 0
Mouse>
rotate the molecule with the left mouse button so that the
green strands are at the top and the large arginine side-
chain of the inhibitor is at the left
Another random document with
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They are living in a new hotel and are most comfortably lodged. They
pay a rouble a day for a room. Their rooms are far more comfortable
and much cleaner than mine. We had beer, vodka, cucumbers,
sardines and cold sausage, and we discussed very many subjects.
During the afternoon many other members dropped in, and among
them a member of the “Council of Empire.” These peasants, who
come from an exceedingly distant government, belong to the more
educated category. I believe the education in their particular
government is good owing to the energy the Zemstva have displayed
there. There are three of these peasants: one of them is a sensible
man who does not know much about things outside Russia; but one
of the others is quite well acquainted with the main features of
European politics and talks of Jaurés, Chamberlain, and Lord
Rosebery. “Who would have thought two years ago,” said one of
them, “that we should see an Englishman here in the flesh?”
July 8th.
July 9th.
July 11th.
July 12th.
The Bill which the Duma passed last week abolishing capital
punishment was discussed in the Upper House the day before
yesterday and referred to a Committee. As the treatment of this
matter has excited no little bewilderment abroad, it will, perhaps, not
be useless to go further into the history of capital punishment in
Russia, which I have mentioned in a previous letter. Capital
punishment was abolished in Russia by the Empress Elizabeth, the
daughter of Peter the Great, in 1753. But as long as the knout was in
use it was rather the name of the thing than the thing itself which
was abolished, because a hundred lashes of the knout meant death.
During the last years in which the knout was employed the number
of lashes was limited to thirty-five. Its use was abolished by the
Emperor Nicholas in the first year of his reign (1825). Beating with a
birch was abolished by the Emperor Alexander II. in 1863, except for
peasants; the beating of peasants was abolished in 1904. “Depuis
lors,” writes M. Leroy-Beaulieu in his standard book on Russia, “la
législation Russe est probablement la plus douce de l’Europe.... La
peine capitale a depuis lors été réellement supprimée; à l’inverse de
ce qui se voit en beaucoup d’autres pays, elle n’existe plus que pour
les crimes politiques, pour les attentats contre la vie du Souverain ou
É
la sûreté de l’État.” During almost the whole reign of Alexander II,
from 1855 to 1876, only one man was executed on the scaffold,
namely Karakosof, the perpetrator of the first attempt made on the
Emperor’s life. From 1866 to 1903 only 114 men suffered the penalty
of death throughout the whole of the Russian Empire.
Commenting on these statistics in the Council of Empire, M.
Tagantzef pointed out that, in contradistinction to this, during 1906
up to the month of June, that is, during five months, 108 people have
been condemned to death under martial law, and ninety have been
executed, not counting people who have been killed without a trial.
The cause, therefore, of the present agitation is the fact that capital
punishment exists in Russia for political crimes only by virtue of
martial law. M. Leroy-Beaulieu, in commenting on the first instances
of this turn of affairs, which occurred in 1878, when a political
agitator was executed in Odessa, remarks that a modern State which
abolishes capital punishment should abolish it altogether, “pour ne
point se donner le démenti d’une contradiction rendue parfois
d’autant plus choquante pour la conscience publique qu’il lui
répugne de voir, comme en Russie, le régicide ou le simple
conspirateur politique traité plus sévèrement que le parricide.”
For and against the entire abolition of capital punishment the chief
arguments of each side are at present these: Those who wish capital
punishment to be retained point to the number of political murders
which have occurred during the last year, and especially to the long
list of innocent policemen who have been murdered, and maintain
that if capital punishment is abolished these crimes will increase.
Those who wish it to be abolished say that the existence of capital
punishment, so far from exercising a restraining influence on
political criminals, excites people to murder and makes martyrs of
them. Moreover, they point out that when people expatiate on the
terrible list of political assassinations they altogether overlook their
cause. They are not in all cases the result of irresponsible hysteria.
The defenders of the Government say: “You make martyrs of people
who are merely common murderers;” the opponents answer: “The
Government shuts its eyes to the lawless and criminal acts of its
officials, and the people are obliged to take the law into their own
hands.” This is the present state of the question, and I have
endeavoured to present both sides of it. Quite apart from the political
murders of the last two years, it is interesting to note that, as far as
we can tell, the abolition of capital punishment in Russia has not had
the effect of increasing crime. In 1890 the proportion of homicides
was seven to the million in Russia (7·4), almost exactly the same as
the proportion in the British Isles during that year, which was (7·5).
July 13th.
July 18th.
Things are going badly in the Duma, and there is likely to be a split
among the Cadets on the subject of a proposed Manifesto to the
people, a counterblast to the Ministerial Declaration.
July 19th.
During the last week not only were rumours circulating to the
effect that the resignation of the Ministry had been accepted, but
certain members of the Right positively affirmed that a new Cabinet
taken from the Duma had been formed. It is said now that this task
was offered to M. Shipov, who is the most important representative
of the Moderate parties outside the Duma, and that he refused it.
Now, since yesterday fresh rumours, which have had a bad effect on
the Bourse, are afloat to the effect that all idea of forming a Ministry
from the Duma itself has been abandoned, and that the Government
is contemplating the dissolution of the Duma and the appointment of
a military dictatorship. Whether there is serious foundation for these
rumours I do not know, but it is obvious that there are only four
courses open to the Government:
1. To form a Coalition Ministry under some Liberal leader outside
the Duma.
2. To form a Ministry from the majority of the Duma.
3. To dissolve the Duma.
4. To do nothing.
The Government is said to have tried the first course and to have
failed. The second course it appears to regard as being out of the
question. The third course is said to be under consideration now. The
fourth course answers to the Government’s policy up to the present.
I have talked with several Conservatives lately—not Moderate
Liberals, but Conservatives of the old régime—and their indignation
against the Government was extreme. One of them said that the
formation of a Ministry from the majority of the Duma, namely, the
Cadets, with whom he had no sympathy, was the only chance of
saving the situation; that he could understand the policy of
dissolution; but the Government did neither the one nor the other,
and the people who were paying for this mistake were the landlords
with the destruction and devastation of their property. Another said
to me that there were at present two great dangerous elements in
Russia—the revolutionaries and the Government—and that of the
two the Government was the more dangerous. A third, a large landed
proprietor, said that he preferred to be despoiled by expropriation
rather than to have all his estates devastated and his houses burnt. A
Government taken from the majority of the Duma, he added, was the
only solution, but it should have been done two months ago; now it
was too late. I mentioned the dissolution of the Duma and the
possibility of a dictatorship. “You would want five hundred dictators,
not one,” he answered, “and what is the use of a dictatorship when
the whole country is on fire? The action of the Government has been
like this: it is as if some people had set a town on fire, locked up the
fire engines, and then talked of putting a dictator at the head of the
fire brigade.”
In opposition to this I have heard views expressed which perhaps
reflect those of the Government. One man said to me that it was now
obvious that the Duma, instead of having a pacifying influence, was
merely a cause of disorder; that when it was originally convened he
had believed in its pacifying capacities; but now he was convinced of
the contrary, and the sooner it was dissolved the better. It may be
objected that, though it is after all true that the convening of the
Duma did not pacify the country, it is necessary to reflect under what
conditions it was convened: its hands were tied; the fundamental
laws were altered for this purpose; the Government not only went on
governing as before, but actually took active measures to discredit its
new Parliament at home and abroad. When a Duma was asked for,
the thing meant was Responsible Government. It is over this
question of responsibility that the whole struggle is being carried on.
I have also heard the following argument, which is advanced by the
newspaper Rossia, a semi-official organ, this morning: “What do we
lose by deciding on repressive measures? Even if we fail by giving in
now we should be failing; therefore we are exchanging certain failure
for problematic failure; it is better to give in after a fight than to
surrender without a struggle, and our chances, now that we are
certain of at least one part of the Army, are better than they will be a
year hence, when we shall be certain of nothing. We are told that we
cannot dissolve the Duma without provoking a revolution, but, from
our point of view, to give in to the Duma now is equivalent to
sanctioning a revolution. Let us try and prove that we can dissolve
the Duma, and that they are merely trying to bluff with their threats
of revolution.” The logical result of this policy should be civil war.[3]
3. And it has proved to be civil war; but civil war waged in everyday life and
unaccompanied by an armed rising.
All the revolutionary elements in the country would be
strengthened by a dissolution, and one can safely predict that the
general disorder would be increased. For even now the sporadic
anarchy is increasing daily. Will the dissolution of the Duma relieve
this tension? I think not. The question then suggests itself: Is there
no hope of a peaceful issue?
A Ministry formed from the majority of the Duma is the only hope;
but whether it would manage to calm matters is another question. It
is true that there is a moderate element, especially among the
peasants, who wish to meet the landlords halfway, who consider the
demands of the Extreme Left, and especially their agrarian
programme, to be absurd. These men would support a Ministry
taken from the Duma, but they continually assert that the
Government will not meet them half way, and that, on the other
hand, they consider the schemes which the Government have put
forward to be fundamentally insufficient. Whether a Ministry
composed of members taken from the majority of the Duma would
succeed in calming the country depends on the nature and intensity
of the opposition they would have to encounter, which it is
impossible to gauge at present. One thing is certain, that in the event
of such a Ministry being given a free hand sympathy would cease to
be extended to the throwers of bombs, whose task is now greatly
facilitated by the simple fact that popular opinion is with them.
When people, on the other hand, say that the Cadets have no men
with whom to form a Ministry—and, to be fair, I have only heard this
argument advanced either in England or by some Russian officials
here—I have heard it contradicted by intelligent Russian officials—
they are talking egregious nonsense. People like Professor Miliukov,
MM. Nabokor, Kokoskin, Muromtzeff, and Petradjinski have shown
themselves not only to be men with brains but to possess political
capacity and tactical ability of no mean order. Even if they were
twenty times less capable than they are they would be more capable
of governing the country than the present Ministry. But
unfortunately it does not seem probable that they will ever win the
confidence of the Crown, since most of them in the past have
suffered for their political principles, and some of them have been in
prison. Therefore, whereas if they had been born in France or
England they would by now be occupying exalted positions, they are
now looked upon from above as men of the same category as
Anarchists and throwers of vitriol. If Mr. Balfour had been born in
Russia he would certainly have been requested to confine his
energies to golf and metaphysics, but if Mr. Haldane had been born
here he would have probably been sent to think about the path to
reality in the paths of Transbaikalia. Therefore at the root of the
whole matter there is a great misunderstanding between the Crown
and the Duma. It is based on the supposition that the Duma is not
representative, and that the revolution is an artificial thing.
July 20th.
July 21st.
July 23rd.
Later.
News has come of the appeal the ex-members of the Duma have
made to the country, urging citizens not to pay taxes and to refuse to
serve in the Army. Everybody is agreed that their action is a fatal
mistake, since they have no means of having any such measures
carried out.
CHAPTER XXV
IN THE COUNTRY AFTER THE
DISSOLUTION
I have been staying for the last three days in the country quite close
to Moscow. I thought I should get away for a time from politics, from
talk of new Cabinets, new eras, liberal autocracy, strong-handed
reform, and other such pleasing illusions. I was mistaken. Politics
filter through everywhere now; in a third-class railway carriage, at
the station buffets, in the public parks, in the villages.
As regards the various opinions I heard expressed the prevailing
one is this: that the new Prime Minister’s programme of strong-
handed Liberal reform is a repetition of the programme of the last
five Ministers of the Interior.
M. Stolypin says these last five Premiers were all mistaken in their
policy; in the meantime (people say) it is difficult to see in what
respects his programme is to differ from theirs. And we have no
evidence as yet that M. Stolypin is an infinitely more capable man
than Count Witte. Some people, referring to the official denial of the
article that appeared in the semi-official newspaper Rossia, with
regard to foreign intervention, say: “If M. Stolypin cannot control the
first page of his official newspaper, how can he expect to control
Russia?” Others commenting on his intention to initiate social
reform and put a stop to the political movement, say that this effort
is the very root and kernel of the whole trouble in Russia; that the
mistake of would-be reformers has always consisted in their not
understanding that social reforms are impossible unless they are
preceded by political reforms. (M. Leroy-Beaulieu, in his splendid
book on Russia, writes in a most illuminating fashion on this very
point.)
As regards what is actually happening in Moscow, the town is
empty and quiet; public meetings are forbidden, small political
gatherings in private houses are held only under surveillance of the
police; gatherings of the “Black Gang” are said to be allowed; the
Press is certainly subjected to a rigid censorship; the Morning Post
arrived blacked out yesterday for the first time for two years; the
manifesto of the ex-members is being spread, likewise the manifesto
of the Social Democrats. I have not seen anybody who thinks that an
era of peace and resigned content has begun.
Near the house where I am living there is a village; as this village is
so close to the town of Moscow I thought that its inhabitants would
be suburban, and therefore not representative of peasant life. This is
not so. The nearness to Moscow seems to make no difference at all. I
was walking through the village on Saturday morning when a
peasant who was sitting on his doorstep called me and asked me if I
would like to eat an apple. I accepted his invitation. He said he
presumed I was living with X., as other Englishmen had lived there
before. Then he asked abruptly, “Is Marie Alexandrovna in your
place?” I said my hostess’s name was Marie Karlovna. “Of course,” he
said, “I don’t mean here, but in your place, in your country.” I didn’t
understand. Then he said it again very loud, and asked if I was deaf. I
said I wasn’t deaf and that I understood what he said, but I did not
know to whom he was alluding. “Talking to you,” he said, “is like
talking to a Tartar. You look at one and don’t understand what one
says.” Then it suddenly flashed on me that he was alluding to the
Queen of England. “You mean Queen Alexandra,” I said, “the sister
of the Empress Marie Feodorovna.” “That’s what I mean,” he said. It
afterwards appeared that he considered that England had been semi-
Russianised owing to this relationship; he thought of course that
both the Queen and the Empress were Russians.
Two more peasants joined us, and one of them brought a small
bottle (the size of a sample) of vodka and a plate of cherries. “We will
go and drink this in the orchard,” they said. So we went to the
orchard. “You have come here to learn,” said the first peasant, a
bearded man, whose name was Feodor. “Many Englishmen have
been here to learn. I taught one all the words that we use.” I said I
was a correspondent; that I had just arrived from St. Petersburg,
where I had attended the sittings of the Duma. “What about the
Duma?” asked the other peasant. “They’ve sent it away. Will there be
another one?” I said a manifesto spoke of a new one. “Yes,” said
Feodor, “there is a manifesto abolishing punishments.” I said I
hadn’t observed that clause. “Will they give us back our land?” asked
Feodor. “All the land here belongs to us really.” Then followed a long
explanation as to why the land belonged to them. It is the property,
as a matter of fact, of the Crown. I said I did not know. “If they don’t
give it back to us we shall take it,” he said simply. Then one of the
other peasants added, “Those manifestoes are not written by the
Emperor but by the ‘authorities.’” (The same thing was said to me by
a cabman at St. Petersburg, his reason being that the Emperor would
say “I,” whereas the manifesto said “We.”) Then they asked me why
they had not won the war; and whether it was true that the war had
been badly managed. “We know nothing,” he said. “What newspaper
tells the truth? Where can we find the real truth? Is it to be found in
the Russkoe Slovo?” (a big Moscow newspaper). They asked me
about the Baltic Fleet and why Admiral Nebogatoff had hoisted a
signal which meant “Beat us.”
Then I went away, and as I was going Feodor asked me if I would
like to go and see the haymaking the next day. If so I had better be at
his house at three o’clock in the afternoon. The next day, Sunday, I
kept my appointment, but found nobody at home in the house of
Feodor except a small child. “Is Feodor at home?” I asked. Then a
man appeared from a neighbouring cottage and said: “Feodor is in
the inn—drunk.” “Is he going to the haymaking?” I asked. “Of course
he’s going.” “Is he very drunk?” I asked. “No, not very; I will tell him
you are here.” And the man went to fetch him. Then a third person
arrived, a young peasant in his Sunday clothes, and asked me where I
was going. I said I was going to make hay. “Do you know how to?” he
asked. I said I didn’t. “I see,” he said, “you are just going to amuse
yourself. I advise you not to go. They will be drunk, and there might
be unpleasantness.”
Then Feodor arrived, apparently perfectly sober except that he was
rather red in the face. He harnessed his horse to a cart. “Would I
mind not wearing my hat but one of his?” he asked. I said I didn’t
mind, and he lent me a dark blue yachting cap, which is what the
peasants wear all over Russia. My shirt was all right. I had got on a
loose Russian shirt without a collar. He explained that it would look
odd to be seen with some one wearing such a hat as I had. It was a
felt hat. The little boy who was running about the house was Feodor’s
son. He was barefooted, and one of his feet was bound up. I asked
what was the matter with it. The bandage was at once taken off and I
was shown the remains of a large blister and gathering. “It’s been
cured now,” Feodor said. “It was a huge blister. It was cured by
witchcraft. I took him to the Wise Woman and she put something on
it and said a few words and the pain stopped, and it got quite well.
Doctors are no good; they only cut one about. I was kicked by a horse
and the pain was terrible. I drank a lot of vodka and it did no good;
then I went to the Wise Woman and she put ointment on the place
and she spoke away the pain. We think it’s best to be cured like this—
village fashion.” I knew this practice existed, but it was curious to
find it so near Moscow. It was like finding witchcraft at Surbiton.
Then we started for the hay meadows, which were about ten miles
distant. On the road we met other peasants in carts bound for the
same destination. They all gravely took off their hats to each other.
After an hour and a half’s drive we arrived at the Moscow River, on
the bank of which there is a tea-shop. Tea-shops exist all over Russia.
The feature of them is that you cannot buy spirits there. We stopped
and had tea. Everybody was brought a small teapot for tea and a
huge teapot of boiling water, and very small cups, and everybody
drank about four or five cups out of the saucer. They eat the sugar
separately, and do not put it into the cup.
Then we crossed the river on a floating bridge, and driving past a
large white Byzantine monastery arrived at the green hay meadows
on the farther river bank towards sunset. Then the haymaking began.
The first step which was taken was for vodka bottles to be produced
and for everybody to drink vodka out of a cup. Then there was a great
deal of shouting and an immense amount of abuse. “It doesn’t mean
anything,” Feodor said. “We curse each other and make it up
afterwards.” Then they drew lots for the particular strip they should
mow; each man carrying his scythe high over his shoulder. (“Don’t
come too near,” said Feodor; “when men have taken drink they are
careless with scythes.”)