X-ray Crystallography

Piotr Sliz
BCMP 201
sliz@crystal.harvard.edu
Computer demos:

Symmetry

Maps
Handouts:

Wave Functions

Symmetry
Reading

Crystallography Made Crystal

Clear, Gale Rhodes
Software

HKL2000 - data processing

molrep/BnP - phasing

CNS - renement

COOT - model building

Ar
Gl
structure
O
O
O
AcHN
OH
O
peptapeptide
O
O
HO
AcHN
OH
O
O
O
AcHN
OH
O
peptapeptide
O
P
O
O
O
P
O
O
OLipid
O
HO
HO
AcHN
OH
O
O
O
AcHN
OH
O
peptapeptide
O
P
O
O
O
P
O
O OLipid
TG
Membrane
Growing peptidoglycan chain
Donor
Lipid II
Acceptor
Model 1
reducing end
elongated chain
Gal
non-reducing end
drug design
mechanism
biology
forceelds
and folding
http://cmcd.med.harvard.edu
Quo Vadis
Structural Biology?
5 - 6 MAY | 2008
SPEAKERS:
Axel Brunger
Naomi Chayen
James Chou
Frank Delaglio
Ben Eisenbraun
Paul Emsley
Joachim Frank
Rachelle Gaudet
Mark Gerstein
David Gohara
Nikolaus Grigorieff
Ian Levesque
Stephen C Harrison
Ralf Grosse-Kunstleve
Miron Livny
Gal McGill
Harry Powell
Stefan Raunser
Jason Schnell
David E Shaw
Piotr Sliz
Woody Sherman
Ian Stokes-Rees
NANOCOURSES:
(symposium registration is required)
Advanced Crystallography
Structure-based Lead Discovery*
Roadmaps for Structure Determination
(EM, NMR, XRAY)
Animating your data*
Mac OSX Development*
Intro to Python*
Bioinformatics*
Sys admin for structural biologists*
Graduate students from Harvard for credit,
all others IDB certificate.
Organized in Mac Classrooms
(75 workstations provided by Apple.)
Early Registration Deadline:
February 29th, 2008
Registration assistance:
www.sbgrid.org/quovadis
The symposium will focus on computational methods in
biology. Data animation, molecular simulation, introduc-
tory programming and methods in structure determination
will be covered by a diverse group of lecturers.
In addition we will review trends in structural biology and
share perspectives on its future direction.
Organizers:
Dr. Piotr Sliz (Chair)
SBGrid, Center for Molecular
& Cellular Dynamics
Harvard Medical School
sliz@crystal.harvard.edu
Dr. Meg Bentley (Vice-Chair)
iDB Educational Initiative
Harvard Medical School
meg_bentley@hms.harvard.edu
Harvard Medical School
BOSTON
*
Stanford
Imperial College
Harvard
NIH
SBGrid
Oxford
Wadsworth
Harvard
Yale
Washington U
Brandeis
SBGrid
Harvard
Berkley Lab
U Wisconsin
Digizyme
MRC
Harvard
Harvard
David E Shaw
SBGrid
Schrdinger
SBGrid
www.sbgrid.org/quovadis
May 5 and 6th
meg_bentley@hms.harvard.edu
Phase IA
1965 Phillips -determined the first 3-d structure of an
enzyme; lysozyme. Second protein structure to be solved.
See Nat.struc. Biol. (Nov. 1998). 5(11). pp 942-926. for a
more detailed history.
Phase IB
1971-74 Application of synchrotron radiation to a
protein crystallography: Rosenbaum, G. Homles, K.C. &
Witz, J. Wychoff & Harrison Phillips, J.C. Wlodawer, A.
Yevitz, M.M & Hodgson.
Phase IIA
1988 Guss et al -first structure solved by the MAD
method/Introduction of second generation synchrotron
sources.
Phase IIB
1990 Teng describes a method of collecting data from a
single protein crystal mounted in loops by cryofreezing.
[Tsu-Yi Teng (1990) j. Appl. Cryst. 23. 387-391.
Phase IIIA
1999 Rosenbaum et al. Third generation protein beam
lines
Phase IIIB
2003 better understanding of radiation damage/RI P?.
Macromolecular X-ray crystallography
(proteins, viruses, DNA, RNA)
1. Crystals
2. Structure Determination
a. From crystals to diffraction
b. From diffraction to electron density
c. From electron density to models
3. Structure quality and statistics
reciprocal lattice electron density map crystal model
Crystal lattice:
Unit Cell: the portion of a space lattice that is repeated in order to form the entire lattice
Asymmetric Unit: the smallest structural unit which, when operated upon by the
symmetry elements of the space group, yields the total crystal structure.
Rotational symmetry
n=2
n=3
n=4
n=6
Rotational symmetry of order n, also called n-fold rotational symmetry, or discrete rotational symmetry of nth order, with
respect to a particular point (in 2D) or axis (in 3D) means that rotation by an angle of 360/n (180, 120, 90, 60.) does not
change the object.
monoclinic
tetragonal cubic
trigonal
cubic
hexagonal
32
622
422
4
432
triclinic orthorhombic
Crystal Systems
2 222 1
monoclinic

A screw axis is a symmetry operation describing how a combination of rotation about an axis and
a translation parallel to that axis leaves a crystal unchanged.

If = 360/n for some positive integer n, then screw axis symmetry implies translational symmetry
with a translation vector which is n times that of the screw operation.

The possibilities are 21, 31, 41, 42, 61, 62, and 63, and the enantiomorphous 32, 43, 64, and 65.
Screw Axis Symmetry and Point Groups
Tables:
P2
1
2
1
2
1
: Origin at
midpoint off three non-
intersecting pairs of
parallel 2
1
axes.
P2
1
: Origin on 2
1
.
Tables:
Protein Crystallization
Full Factorial Incomplete Factorial
Random Sparse Matrix
All elements of the matrix
of parameters are sampled.
Factor levels are chosen randomly and
then balanced to achieve uniform
sampling. All two-factor interactions
are sampled as uniformly as possible.
Random sampling of all parameters, but it
approximates incomplete factorial designs.
Intentional bias towards combinations of
conditions that have worked previously.
Crystallization screens
Rational Screens for Protein Crystallization:
Microuidics can provide a robust and
systematic environment for crystallization
screening.
Protein solubility prole
Microuidics device.
Macromolecular X-ray crystallography
(proteins, viruses, DNA, RNA)
1. Crystals
2. Structure Determination
a. From crystals to diffraction
b. From diffraction to electron density
c. From electron density to models
3. Structure quality and statistics
reciprocal lattice electron density map crystal model
Images of Microscopic Objects
d = "/(2n sin#)
minimum
separation
wavelength
The minimum separation (d) that can be resolved by any
kind of a microscope is given by the following formula:
Magnication:
Lens Optics (wikipedia):
Obtaining Images of Molecules
Wavelength must be
corresponding to the object.
X-rays (~10
-10
m or 1 angstrom)
Suitable radiation:
Crystallographic Analogy of Lens Action:
Braggs Law (1/2):
Constructive interference: distance traveled by two waves differs
by a multiple of a wavelength.
Lattice planes reect X-rays.
x
y 010
110
100
210 310
110
Braggs Law (2/2):
A
B
C
Difference in path: 2xBC:
Reciprocal Space:
Whenever the crystal is rotated so that a reciprocal-lattice point comes
in contact with the circle of radius 1/lambda, Braggs law is satised and a
reection occurs.
Direction of reections
and number of reections
depend only on unit-cell
dimensions, and not
contents of the unit cell.
Fig 4.21
The number of measurable reections:
If the sphere of reections has a radius of 1/!, then any reciprocal-lattice point
with a distance of 2/! can be rotated into contact with the sphere of reections.
Total number of measurable reections: number of reciprocal unit cells within the limiting sphere.
Number of required reections is limited by diffraction resolution and symmetry.
Wave equation:
f(x) = Fcos2!(hx + ")
vertical height at any
horizontal position x along
the wave
amplitude
frequency
phase
any simple wave function can be
described in three constants:
Simple wave functions: f(x) = Fcos2!(hx + ")
f(x) = 3cos2!(x)
f(x) = cos2!(x) f(x) = cos2!(5x)
f(x) = cos2!(x+1/4)
Complicated wave functions:
Complicated periodic functions can be described as the
sum of simple sine and cosine functions.
Fourier terms
Fourier
series
Each Fourier term is a simple sin or cos function.
simple wave
complex number wave (a + i b)
Fourier Series:
unspecied number of waveforms
Fourier Series
(exponential form):
equality from complex
number theory:
Fh - intensity
h - frequency
simple
diffraction
waves
Fourier
Synthesis
Reciprocal
Space
Real
Space
Fourier series for each reection is a
sum of contributions from individual
atoms in the unit cell.
Each reection is a contribution from all atoms in the unit cell.
The structure factor is a wave created by the superposition of many individual waves (described as Fourier series).
Fhkl= fA+fB+...+fA+FB+..+ fF
Structure-factor equation:
Data Collection:
1) INDEX (determine unit cell and space group)
2) Integrate (measure intensity of reections)
3) Scale (combine partials and scale between frames)
scale, convert I to F
integrate:
structure factor le
Data collection strategy depends
on crystal symmetry
typically 180 1 oscillation frames:
Intensities of systematic absences
h k l Intensity Sigma I/Sigma
0 0 20 6.1 4.8 1.2
0 0 22 -3.5 7.4 -0.5
0 0 23 5.0 5.5 0.9
0 0 25 -3.8 8.6 -0.4
0 0 26 9.1 6.9 1.3
Data Collection Statistics:
overall/high res bin
Rules of three to determine resolution
limit (calculated in high res bin):
1. Completeness > 70%
2. Rsymm < 30%
3. I/sigma > 3
orthorhombic (4 molecules / unit cell)
mosaicity (workable range: 0.1 -1 degree)
resolution model building power
lower than 4 possible to t exist structures (rigid body renement)
3.3-4 limited remodeling of existing structures
2.5 - 3 de novo model building
higher than 2.5
atomic details visible (model waters, detailed hydrogen
bonding network, alternative conformations)
Summary of reflections intensities and R-factors by shells
R linear = SUM ( ABS(I - <I>)) / SUM (I)
R square = SUM ( (I - <I>) ** 2) / SUM (I ** 2)
Chi**2 = SUM ( (I - <I>) ** 2) / (Error ** 2 * N /
(N-1) ) )
In all sums single measurements are excluded
Shell Lower Upper Average Average Norm. Linear Square
limit Angstrom I error stat. Chi**2 R-fac R-fac
30.00 4.31 1521.8 30.8 19.4 15.312 0.108 0.150
4.31 3.42 1753.0 36.3 23.8 16.596 0.118 0.160
3.42 2.99 866.4 20.6 15.6 13.148 0.130 0.168
2.99 2.71 506.4 14.3 11.8 10.336 0.142 0.186
2.71 2.52 363.8 12.1 10.5 8.596 0.150 0.195
2.52 2.37 298.4 11.1 9.9 7.195 0.156 0.202
2.37 2.25 228.2 10.1 9.3 5.693 0.160 0.198
2.25 2.15 188.1 9.6 9.0 5.098 0.171 0.213
2.15 2.07 150.3 9.1 8.7 3.907 0.175 0.207
2.07 2.00 122.2 8.8 8.5 3.186 0.184 0.214
All reflections 605.3 16.4 12.7 8.857 0.130 0.162
Shell Summary of observation redundancies:
Lower Upper % of reflections with given No. of observations
limit limit 0 1 2 3 4 5-6 7-8 9-12 13-19 >19 total
30.00 4.31 7.5 4.2 10.3 17.8 19.3 23.9 11.3 5.7 0.0 0.0 92.5
4.31 3.42 2.1 3.8 9.9 23.0 22.5 20.3 13.5 4.8 0.0 0.0 97.9
3.42 2.99 0.8 2.8 6.6 20.6 25.6 27.8 12.7 3.1 0.0 0.0 99.2
2.99 2.71 0.6 1.8 5.3 21.8 25.4 29.6 13.0 2.5 0.0 0.0 99.4
2.71 2.52 0.2 1.4 5.0 21.1 25.5 32.2 12.2 2.4 0.0 0.0 99.8
2.52 2.37 0.0 0.8 4.5 21.4 26.7 32.3 12.3 2.0 0.0 0.0 100.0
2.37 2.25 0.0 0.6 4.3 20.0 27.8 33.7 11.9 1.7 0.0 0.0 100.0
2.25 2.15 0.0 0.3 3.9 21.0 27.9 34.7 10.7 1.5 0.0 0.0 100.0
2.15 2.07 0.0 0.4 3.0 20.9 27.2 36.3 10.3 1.9 0.0 0.0 100.0
2.07 2.00 0.0 0.3 3.4 20.0 28.1 36.9 9.1 2.1 0.0 0.0 100.0
All hkl 1.2 1.7 5.7 20.7 25.5 30.6 11.7 2.8 0.0 0.0 98.8
Macromolecular X-ray crystallography
(proteins, viruses, DNA, RNA)
1. Crystals
2. Structure Determination
a. From crystals to diffraction
b. From diffraction to electron density
c. From electron density to models
3. Structure quality and statistics
reciprocal lattice electron density map crystal model
Fourier series for electron density is
a sum of contributions from
individual reections.
simple
diffraction
waves
Fourier
Synthesis
Fourier
Analysis
Fourier Transform:
Reciprocal
Space
Real
Space
Phase Problem:

amplitudes (can be measured,

~ sq rt of intensity)

frequency (X-ray source)

phase ??
F
hkl
F
Real
Real
Solving Phase Problem: Molecular Replacement
1) Rotation Function 2) Translation Function
Combining model phases with experimental intensities will
reveal details of missing elements.
Typically 30% identity and 1/3 of a structure required.
Homologous
or incomplete
model:
Self Rotation Function
MOLREP
Rad : 30.00 Resmax : 3.00 RF(theta,phi,chi)_max : 0.1137E+05 rms : 771.3
Chi = 180.0
X
Y
RFmax = 0.1137E+05
Chi = 90.0
X
Y
RFmax = 1254.
Chi = 120.0
X
Y
RFmax = 1038.
Chi = 60.0
X
Y
RFmax = 1038.
Polar angles theta, phi, chi dene the
standard system orientation in the cell.
Theta, phi - polar coordinates of Z standard
axis. Chi - angle of rotation around theta-
phi-axis (Z standard axis) which bring X
axis to standard X axis.
Number of RF peaks : 10
theta phi chi alpha beta gamma Rf Rf/sigma
Sol_RF 1 146.34 -139.17 155.19 55.63 65.55 153.97 0.1453E+07 3.69
Sol_RF 2 132.29 165.77 54.08 56.82 39.30 265.27 0.1409E+07 3.57
Sol_RF 3 126.82 159.77 57.42 51.60 45.23 272.06 0.1395E+07 3.54
Sol_RF 4 147.99 -141.48 160.47 49.99 62.99 152.96 0.1387E+07 3.52
Sol_RF 5 154.58 -138.22 162.21 51.61 50.18 148.05 0.1285E+07 3.26
Sol_RF 6 127.83 148.20 69.52 35.15 53.52 278.74 0.1265E+07 3.21
Sol_RF 7 43.33 96.64 95.04 45.10 60.81 31.83 0.1252E+07 3.18
Sol_RF 8 61.94 104.17 98.84 42.94 84.17 14.61 0.1213E+07 3.08
Sol_RF 9 82.25 169.61 53.05 83.46 52.52 284.24 0.1213E+07 3.08
Sol_RF 10 24.69 44.74 106.19 5.17 39.02 95.68 0.1185E+07 3.01
3
2
2
1
1
Finding heavy atom sites using Patterson methods:
-Fh+Fph=Fp
110
310
Harker Construction:
Macromolecular X-ray crystallography
(proteins, viruses, DNA, RNA)
1. Crystals
2. Structure Determination
a. From crystals to diffraction
b. From diffraction to electron density
c. From electron density to models
3. Structure quality and statistics
reciprocal lattice electron density map crystal model
rebuilding
renement:
real
space
reciprocal
space
annealing
rigid body
minimization
b-factor
Macromolecular X-ray crystallography
(proteins, viruses, DNA, RNA)
1. Crystals
2. Structure Determination
a. From crystals to diffraction
b. From diffraction to electron density
c. From electron density to models
3. Structure quality and statistics
reciprocal lattice electron density map crystal model
Kleywegt et al. Homo crystallographicus--quo vadis?. Structure (2002) vol. 10 (4) pp. 465-72
R
free
10 x resolution