LOSCHMIDT
LABORATORIES
Structure of biomolecules
□ Proteins
Primary structure Secondary structure Tertiary structure Motifs and folds Quaternary structure
□ Nucleic acids
■   Main types of structures
□ Primary structural databases
□ Structural data formats
□  PDB and mmCIF formats
Structure of biomolecules
Primary structure
Secondary structure
Lys		
Lys		
Gly		
Gly 1-■-1		
Leu -^-		
Val		4^—J
Ala		
His		
Tertiary structure
Quaternary structure
Amino acid residues
a Helix
Polypeptide chain
Assembled subunits
Proteins - hierarchy of protein structure
□ 20 L-amino acids (natural)
□ Side chains
■   Charged, polar, hydrophobic
A.   Amino Acids with Electrically Charged Side Chains
Positive
Arginine (Arg) O
Amino acid backbone
Histidine (His) (J)
NH
-s
Lysine
(Lys)Q
NH,
NH,
B.   Amino Acids with Polar Uncharged Side Chains
Serine	Threonine	Asparagi	ie Glutamine
(Ser)Q	(Thr)Q	(A»n) 0	(Gln)0
			
			
)-NH2	)-NH2	>	-NH2 )—NH
(	HO—(	<	<
\ OH	\	>	° _)
		NH2	
Negative
_A_
S- -\
Aspartic Acid Glutamic Acid (Asp)0 (Glu)Q
NH,
NH,
C.   Special Cases
Side . chain
Cysteine	Glycine	Proline
(Cys) 0	(Gly) 0	(Pro)Q
"P		HP
		
\-NH2	\_	-NH2 V
\ SH		s
Amino group
HoN
'a
t
Chiral centre
COOH \
Acid group
D.   Amino Acids with Hydrophobic Side Chains
Alanine Valine Isoleucine
(Ala) Q (Val)Q dl') O
Leucine (Leu)0
0=\
o=(
NH.
Methionine Phenylalanine Tyrosine
(Met)(J) (Ph.)Q (Tyr)Q
Tryptophan (Trp) ©
NH,
□ Linear chain of amino acid residues
MSLGAKPFGEKKFIEIKGRRMAYIDEGTGDPILFQHGNPTSSYLWRPJIM
N-terminus
C-terminus
r
□ Protein backbone
■ From N-terminus to C-terminus
■ Connected by covalent bonds
□ Peptide bond (amide bond)
Partial double bond character -> Planar geometry
RiV 0 R2 k,^-;
+ .       i <—> + i ^ i r
Hj+N—CH—C-N—CH—COO" H3 N—CH—C=N—CH—COO"
v.
H2N-C — C
Amino
H
I
C I
\
OH Acid
Amino
H
l ^
R2
Acid
O OH
condensation -H20
Amino    Acid Amino
Proteins - primary structure
Geometry of protein backbone
□ Conformation of the peptide chain
■   Defined by O (phi) and ^ (psi) dihedral angle
□ Ramachandran plot (0, 4J)
-> The majority of proteins follow this distribution
180
■180
	%
	
Amide plane
Side group
Amide plane
■180
180
cp (phi) = dihedral angle {C - N - Ca - C} Y (psi) = dihedral angle {N - Ca - C - N}
Proteins - primary structure
□ Local three-dimensional structure of polypeptide chain
□ Governed by hydrogen bonding between backbone
atoms
□ DSSP (hydrogen bond estimation algorithm)
■ The most common method for assigning secondary structure
■ Starts by identifying the intra-backbone hydrogen bonds
(between NH.....0=C)
■ Hydrogen bond exists if E < -0.5 kcal/mol
■ The type of repetition will assign the residue to one of 7 types
(3 major types: helices, strands and loops)
E = 0.084 { —+ —-------— 1 - 332 kcal/mol
L roN     rcH     r0H     ^CN J
Polypeptide bond
Proteins - secondary structure
□ Types of helices
■ 3.613 helix (a-helix) - most common
■ 310 helix - less frequent, end of a-helices
■ 4.116 helix (n-helix) (rare)
■ Left-handed helix (very rare) -> Represented by helical
cartoons or cylinders
IP
□ Right-handed (mostly)
□ Hydrogen bonding
■   Within a single chain
310-helix ff* ^	Left-handed a-helix (
R-helix      . .. a-hehx	
Proteins - secondary structure
Type	3M	a	71
			
Residues per turn	3.0	3.6	4.1
Atoms in H-bonded ring	10	13	16
Hydrogen bonding	n - n + 3 n	— 71 +	4           77 - n + 5
Angle between neighboring residues	120	100	88
Helical rise per amino acid residue (A)	2.0	1.5	1.15
	-75	-60	-75
	-5	-45	-40
□ Types of typical ß-structures
■ ß-sheets
■ ß-turns
■ ß-bulge
■ Polyproline helices
□ Hydrogen bonding ^
■   Between adjacent chains
ß-sheets	1 polyproline helicei
	
Proteins - secondary structure
the sheets
Proteins - secondary structure
□ (3-turns
■ Short structures (4-5 residues)
■ Connects two (3-strands
■ Ideally H-bond between backbone of n and n+3 residues
■ Often includes glycine or proline on specific positions
□ (3-bulge
■ Frequently occurs in antiparallel (3-sheets
■ Disrupts ideal H-bonding pattern
■ Increases twists of a sheet
□ Polyproline helices
■ Typical in collagen and other strong fibers
■ Left-handed triple-stranded helix (unlike most of other helices)
■ Composed of three chains of repetitive sequence (Proline-
□ Global three-dimensional structure of protein
□ Governed mainly by hydrophobic interactions involving
□ Supersecondary structures (motifs)
■ Small substructures formed by several secondary structures
□ Domain
■ Structurally (functionally) independent regions
■ Compact parts of structure - around single hydrophobic core
■ Formed in separate folding unit (fold independently)
□ Fold
■ General architecture of protein
■ Type of protein structure
□ ß-harpin
□ ß-meander
□ Greek key
□ Jellyroll
Proteins - tertiary structure
□ Helix-turn-helix
□ Helix bundle
□ ßaß unit
Proteins - tertiary structure
□ Parts of tertiary structure
■ Separate folding
■ Independent structure
■ Usually up to 200 residues
Pyruvate kinase in glycolysis
pyruvate kinase
ADP
ATP
PEP
-> pyruvate
Nucleotide binding
Substrate
binding
Regulatory domain
Proteins - tertiary structure
□ Some folds are very common, some are rare
□ Classification of folds
■ Biochemical
■   Globular, membrane, fibrous proteins, intrinsically disordered
■ Structural
□ Number of folds
Theoretical maximum: 10,000
Currently: 1,195 (SCOP) vs 1,373 (CATH)
all-a, all-ß, a/ß and a+ß proteins
Proteins - tertiary structure
□ Globular proteins
□ Membrane proteins
□ Fibrous proteins
Structural classification of folds
□ All-a (entirely a-helices)
Up-and-down bundle
Globin-like
□ All-ß (entirely ß-strands)
Jellyroll
ß barrel
ß propeller
Proteins - tertiary structure
□ a/ß (sequence alternates between a-helices and ß-strands)
Rossmann
TIM barrel
□ a+ß (a-helices and ß-strands occur separately in sequence)
ß-Grasp (ubiquitin-like)
Proteins - tertiary structure
□ Association of several protein chains (monomers/subunits) into oligomers (multimers)
■ Homomeric protein - from identical monomers
■ Heteromeric protein - from different types of monomers
Homotetramer hemoglobin
Heterodimer tryptophan synthase
Heterotetramer immunoglobin
Proteins - quaternary structure
□ Composition
Nucleotide
Phosphate group is bonded to 5' carbon of sugar
o
II i -o—p—o
f—u —ia o r Phosphate
N Nitrogenous base
group
©charge
Sugar
ch2
*c' h hc1
I \'_■ /1
h 3<f—h oh oh
Ribose in RNA
3 2
5-carbon suaar
4
Nitrogenous base is bonded to 1' carbon of sugar
"?H2 ^o.
"c^h    h   c' h ,c—c2, h oh h Deoxyribose in DNA
Nitrogenous base
o
nh,
o
□ Phosphate
□ Pentose sugar
Cytosine(C) Uracil (U) in RNA     Thymine (T) in DNA □ HeterOCyClJC b3Se
Pyrimidines
]
^^l^^nh
vn^^n^nh2
J
Guanine (G)
nh2
j
Adenine (A)
Purines are larger than pyrimidines
□ DNA bases: A, G; T, C
□ RNA bases: A, G; U, C
Purines
]
□ Rotation about glycosidic bond
NH,
CO
HO
0-.
HO
u3
%|^N
HO
NH2 N
N^O
O-
OH
anti
oh
syn
OH
anti
The anti conformation is dominant in DNA with rare exceptions
Nucleic acids - basic building blocks
□ Linear chain of nucleotides (oligonucleotides or polynucleotides)
[7fccGAATTCGC|G~|
Nucleic acids - primary structure
□ Linear chain of nucleotides (oligonucleotides or
polynucleotides)
[7fccGAATTCGC|G~|
□ Sugar-phosphate backbone
■ Covalent character
■ Phosphodiester bond
■ From 5'-end to 3'-end
5' end
HO i
0
NH
A,
,0^     N NH2
NH
O
O-P-0
o
2 C
N
o
O-P-0
11
o
I NH2 \   J Me
O
O-P-0
0, N
oligonucleotide dGCAT
0
o
NH
±10
3' end
T
Nucleic acids - primary structure
Sugar-phosphate backbone
□ Very flexible backbone
■   Six torsion angles
□ Ribose is not planar -> sugar puckering
■ Denotes the phosphate-phosphate proximity
■ Two main types of conformation
Nucleotide
milt
BASE
C3'-endo (A-Conformation)
BASE
C2'-endo (B-Conformation)
Base
OR H
2'-deoxyribose (in DNA)
Nucleic acids - primary structure
□ Local interactions between nucleotide bases
□ DNA base pairs:
Adenine - Thymine Cytosine - Guanine
□ RNA base pairs:
Adenine - Uracil Cytosine - Guanine
□ Complementarity due to hydrogen bonds
Nucleic acids - secondary structure
□ Leontis/Westhof classification
Three base-paring edges
■ Watson-Crick (WC)
■ Hoogsteen (H)
■ Sugar(S)
12 types of base-paring
4- ^
DO
• wc O
■  H □
► s >
cis trans
^7
Anti-parallel pairs
^
Parallel pairs
Nucleic acids - secondary structure
Tertiary structure of DNA
□ Overall three-dimensional arrangement and folding
□ Three types: A-DNA, B-DNA, Z-DNA
□ B-DNA is the most common (described by Watson & Crick)
A-DNA
(rare)
B-DNA
(predominant) Z-DNA
Type	A-DNA	B-DNA	Z-DNA
Helix sense	Right	Right	Left
Bases per turn	11	10.5	12
Helical rise per nucleotide (A)	2.6	3.4	3.7
Sugar pucker	C3'-endo	C2'-endo	C2'-endo C3'-endo
Nucleic acids - tertiary structure of DNA
□ Grooves: crucial for DNA-protein interactions
□ Major groove: wide and deep - where most proteins interact
BSB BIBB
□ Quaternary structures - with support of proteins
□ Quaternary structures - with support of proteins
Histonc
Acetyl group
(()( H,
B: DNA acetylation
DNA inaccessible, gene inactive
DNA accessible, gene active
Nucleic acids - higher structures of DNA
Secondary structures of RNA
□ Most common form: A-RNA helix (similar to A-DNA)
Nucleic acids - secondary structure of RNA
Secondary structures of RNA
□ Junctions
Regions connecting two or more stems Two-stem, three-stem and four-stem junction
5"
3' -
- 3' 5'
5 3"
F     3 -*J-J-^ ^LJ_L
Nucleic acids - secondary structure of RNA
Secondary structures of RNA
□ Harpin loops
■   Sequence inversely self-complementary GGCUGGCUGUUCGCCAGCC
5' 3'
5' 3"
5' 3'
5' 3"
Many subtypes - e.g.: GNRA, ANYA, UNCG tetraloops
CI4
5" 3'
5' 3'
C91
Nucleic acids - secondary structure of RNA
Secondary structures of RNA
□ Very complex-stem-loop structure
Sz9-
I I I I I ■ - ■ I I I I I I I    Vfi' o
S s
°    I I I I      I ■ I I I I I I
T X    1=1 S
1 'coann^n nn*1 I I I I I
u--t>a
nrjrj
I I I I I I ri^n-njn
•aJ> = °nn_qH if. ■ 1
III *■I I I I I 1. 11    I -1 I I I I
■ j_ ■ '
a— rc-
PI
_o_ n   8   a. =^**7i-
g=|    3ts-s-< a-
I II
5
1 iii' ,_=
1   a—0
I  I I I ■ ■ 1
i^-LuTm!
I I ' -1 ■ ' I I I I       I 11 ■ I ■ I I - •
qZi-^i-jZiZi-^qziCiZiZiZi ^   ^ r«:ZiZirjiTi nrinri^iGO
I I I   I I I    I I     I I I I I   I I I I I I I
*v-^r?«-irT^i>i-i   n-^ nann nrn r.]*nn*r.M
II I I I ■ I I I I ■
if V if "=
Nucleic acids - secondary structure of RNA
Tertiary structures of RNA
A-RNA dodecamer
Phenylalanine transfer RNA
Group I intron ribozyme
Hammerhead ribozyme
Guanine riboswitch
Nucleic acids - tertiary structures of RNA
□ Association of several chains of RNA
Frequently joined with proteins
Eukaryotic ribosome - ~ 6800 nt, 79 proteins
P-tRNA
60S
Growing peptide chain
Outgoing empty tRNA
Incoming tRNA bound to Amino Acid
Ribosome
Peptide Synthesis
Ribosome in action:
https://www.voutube.com/watch?v=Jml8CFBWcDs
Nucleic acids - quaternary structure of RNA
□ Worldwide Protein Data Bank (wwPDB)
http://www.wwpdb.org/
□ RCSB Protein Data Bank (RCSB PDB)
http://pdb.rcsb.org
□ Nucleic Acid Knowledgebase (Nucleic Acid Database)
https://www.nakb.org/
□ Biological Magnetic Resonance Data Bank (BMRB)
https://bmrb.io/
□ Electron Microscopy Data Bank (EMDB)
http://www.emdatabank.org/
□ Cambridge Structural Database (CSD)
http://www.ccdc.cam.ac.uk/products/csd/
W O R L D W IDE
SPDB
P ROTE IN DATA B AN K
SBPDBe
Protein Data Bank in Europe
PROTEIN  DATA BANK
NAKB
Nucleic Acid Knowledgebase
BMRB
Biological. Magnetic Resonance Data Bank
% EMDB
Electron Microscopy Data Bank
...More details in lesson 4!
Primary structural databases
□ Different file formats used to represent 3D structure data
■ PDB
■ mmCIF
■ PDBML
■ MOL2
■
■ ■ ■
□ The spatial 3D coordinates and other information are recorded for each atom
□ Designed in the early 1970s - first entries of PDB database
□ Rigid structure of 80 characters per line, including spaces
□ Still the most widely supported format
Structural data formats - PDB format
structure anrwtation
arrnno acid field
cofactor filed
HEADER	LYASE		(CARBON-CARBON)					03-JUT	-95	1DNP	
TITLE	STRUCTURE OF				DEQJIYPIBODIPYRIHIDINE PBQTOLYASE						
■  *  ■ -hid SOURCE	2 ORGANISM SCIENTIFIC:					ESCHERICHIA COLI					
KEYWDS	UN* REPAIR			, ELECTRON		TRANSFER	,  EXCITATION FHRRGV TRANSFER,				
KE*VJD£	2 LYASE		,  CARBON-CARBON								
ATOM	21	ND1	HIS	A	:i		27.866	62.971	1.00	1 .07	N
ATOM	22	CD2	EilS	A	3	57*200	2B.354	61.B94	1.00	13.12	C
ATOM	23	CEl	HIS	A	2	56.124	26.793	62.991	1.00	13.03	c
ATOM	24	NE 2	HTS	A	3	57,243	27.052	62.334	1.00	a. V)	N
ATOM	25	H	LEU	A	4	55.5B0	32*694	59.656	1.00	12. 61	N
ATOM	26	CA	LEU	A	4	54.799	33.803	59.113	1.00	11.56	c
ATOM	21	C	I.KLI	A	1	53,552	33.265	53.374	1.00	7.76	c
ATOM	28	t:	LEU	A	4	53*650	32.363	57.532	1.00	6.99	0
ATOM	29	CE	LEU	A	4	55.656	34.603	59.174	1.00	9.Q3	c
ATOM	30	CG	LEU	a	1	54.946	35.8B7	57.518	1.00	?A)0	c
ATOM	31	CDl	LEU	A	4	54.623	36.920	56.550		6.2L	c
iir- -MT'.	7641	AN7	FAD	H	472	27.855	76.556	29.073	l . ::o	4.55	N
HETATM	7642	AC 5	FAD		472	2B.524	76.026	27.955	1.00	2.00	c
HETATH	7643	AC 6	FAD	P.	472	2 9.049	77.609	2 7. f24	1.00	3.40	c
BETATM	7iVH	AN 6	FAD	1!	472	3D.7B7	77.757	26.654	1 .::(■	b.s?	N
/
aiorn number
/
residua rsne
residue number
I
\
x. y, z coordinates
occupancy temperature factor
I
atom type
atom
polypeptide chain identifier
Structural data formats - PDB format
□ Atomic coordinates
□ Chemical and biological features
□ Experimental details of the structure determination
□ Structural features
■ Secondary structure assignments
■ Hydrogen bonding
■ Biological assemblies
■ Active sites
■ ■ ■
• https://www.wwpdb.orq/documentation/file-fo^
• https://wwwxql.ucsf.edu/chimera/docs/UsersGuide/tutorials/pdbintro.html
□ Advantages
Widely used -> supported by majority of tools Easy to read and easy to use Can be manually edited
-> Suitable for accessing individual entries
Structural data formats - PDB format
□ Disadvantages
■   Potential inconsistency between individual PDB entries as well as PDB records within one entry Ex: different residue numbering in SEQRES and ATOM sections
-> Not suitable for computer extraction of information
Primary sequence
Atoms and residues in the file
seqres	1	~±9e Qmet			sp glu	asn	ile	thr	ala	ala	pro	ala asp pro	ile
seqres	2	396 leu			gly leu	ala	asp	leu	phe	arg	ala	asp glu arg	pro
atom	1	n	met		5	41	.402	11	.897	15	2 62	1.00  48 .61	
atom	2	ca	met		5	40	919	13	.262	15	600	1.00 47.70	
atom	9	n	phe		6	39	627	14	.840	14	228	1.00  48 .66	
atom	10	ca	phe _		6	39	199	15	.440	12	964	1.00  45 .33	
Structural data formats - PDB format
□ Disadvantages
■   Absolute limits on the size of certain items of data
Ex.: max. number of atom records limited to 99,999; max. number of
chains limited to 26, etc.
-> Large systems such as the ribosomal subunit must be divided into multiple PDB files
-> Not suitable for analysis and comparison of experimental and structural data across the entire database
Structural data formats - PDB format
□ Macromolecular crystallographic information file (mmCIF)
□ Developed to handle increasingly complicated structural data
□ Each field of information is explicitly assigned by a tag and linked to other fields through a special syntax
PDB    HEADER PLANT SEED PROTEIN 1 l-OCT-91 1CBN
mmCIF    _struct. entry_id '1CBM'
_Struct.title   'PLANT  SEED PROTEIN' _struct_keywords.entry_id 11CBN' _struct_keywords.text  'plant seed protein1 _database_2,database_id 'PDB' _database_2,database_code 11CBN' _database_PDB_rev.rev_num 1 database PDB rev,date original '1991-10-11
Structural data formats - mmCIF format
□ Advantages
■ Easily parsable by computer software
■ Consistency of data across the database
-> Suitable for analysis and comparison of experimental and structural data across the entire database
□ Disadvantages
■ Difficult to read
■ Rarely supported by visualization and computational tools
-> Not suitable for accessing individual entries
Structural data formats - mmCIF format
□ Protein Data Bank Markup Language (PDBML)
□ Extensible Markup Language (XML) version of PDB format
<?xml version="l.0" encoding=,,UTF-8" ?> < PDBx:datablock   datablockName="EXAMPLE■
xmlns:PDBx="http://deposit.pdb.org/pdbML/pdbx-vl.000.xsd"
xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"
xsi:schemaLocation="http://deposit.pdb.org/pdbML/pdbx-vl.000.xsd
pdbx-vl.000.xsd"> < PDBx: ent i ty__polyCategory>
<PDBx: ent ity_j>oly entity_id= " 111 >
<PDBx:typepolypeptide(L)</PDBx:type> <PDBx:nstd_linkage >no</PDBx:nstd_linkage > <PDBx:nstd_monomer>no</PDBx:nstd_monomer> <PDBx: pdbx_seg^_one_letter_code> DIVLTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYYTTTLADG VPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPRTFGGGTKLEIK </PDBx:pdbx_seq_one_letter_code> < PDBx:pdbx_seq_one_le11 er_code_can> DIVLTQSPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYYTTTLADG VPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPRTFGGGTKLEIK </PDBx:pdbx_seq_one_letter_code_can> </PDBx:entity_poly> </PDBx:entity_j?olyCategory> </PDBx:datablock>
Structural data formats - PDBML format
□ Gu, J. & Bourne, P. E. (2009). Structural Bioinformatics, 2nd Edition, Wiley-Blackwell, Hoboken.
□ Liljas, A. et al. (2009). Textbook Of Structural Biology, World Scientific Publishing Company, Singapore.
□ Schwede, T. & Peitsch, M. C. (2008). Computational Structural Biology: Methods and Applications, World Scientific Publishing Company, Singapore.
□ Schaeffer, R.D & Daggett, V. (2011). Protein folds and protein folding. Protein Engineering, Design & Selection 24:11-19.