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x-ray Crystallography

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Piotr Sliz

BCMP 201

sliz@crystal.harvard.edu

Computer demos:

Symmetry

Maps

Handouts:

Wave Functions

Symmetry

Reading

Clear, Gale Rhodes

Software

molrep/BnP - phasing

CNS - renement

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:

~ sq rt of intensity)

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

- Atomic Structure and Crystalline Order of Graphene-Supported Ir Nanoparticle Lattices.pdfUploaded byFamiloni Layo
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