Protein structure determination by x-ray crystallography and NMR

Introduction to protein structure

In the last lectures we learned how protein structures are determined experimentally.

Today, we'll focus on how computational techniques are employed to aid

structure determination.


There are two main techniques for solving protein structures: x-ray

crystallography and Nuclear Magnetic Resonance (NMR).

As can be seen from the current

PDB holdings, more than 42,000 protein structures have been solved

so far, and are available from the

Protein Data Bank. About 13% of these are NMR structures, the rest

are x-ray crystallographic structures. As can also be seen on the PDB holdings list,

the number of solved proteins grows even faster, due to advancements

in structure determination techniques. Nevertheless, the number of

known protein sequences is almost an order of magnitude larger

(currently about 276,000, as available from the SWISS-PROT

database).

Reading PDB files

As we've learned last time, protein structures are stored at the Protein Data Bank, from which

individual protein structures can be retrieved as so-called PDB

files. Before we will turn to the structure determination itself, let

us have a closer look at a typical PDB file, to see what can be

learned about the background of the structure (experimental conditions

etc.) and the structural quality (the resolution, coordinate uncertainty).

We will focus on PDB entry 1TRO (an x-ray structure) as it can

be downloaded from the Protein Data bank.

The initial lines of a PDB entry contain information on the

protein, the source, the names of the people who sent the entry to the

PDB, references to literature that describe the structure in detail,

and some basic information regarding the crystallographic

data. Especially critical to check are the resolution and the R-factor

(or free-R factor), that contain information on how well the deposited

structure matches the measured data (x-ray reflection intensities in

this case).


HEADER DNA-BINDING REGULATORY PROTEIN 30-AUG-92 1TRO 1TRO 2

COMPND TRP REPRESSOR COMPLEX WITH OPERATOR 1TRO 3

SOURCE TRP REPRESSOR: (ESCHERICHIA COLI, STRAIN W3110); 1TRO 4

SOURCE 2 OPERATOR: SYNTHETIC 1TRO 5

AUTHOR Z.OTWINOWSKI,R.G.ZHANG,P.B.SIGLER 1TRO 6

REVDAT 1 31-JAN-94 1TRO 0 1TRO 7

REMARK 1 1TRO 8

REMARK 1 REFERENCE 1 1TRO 9

REMARK 1 AUTH Z.OTWINOWSKI,R.W.SCHEVITZ,R.-G.ZHANG,C.L.LAWSON, 1TRO 10

REMARK 1 AUTH 2 A.J.JOACHIMIAK,R.MARMORSTEIN,B.F.LUISI,P.B.SIGLER 1TRO 11

REMARK 1 TITL CRYSTAL STRUCTURE OF TRP REPRESSOR OPERATOR 1TRO 12

REMARK 1 TITL 2 COMPLEX AT ATOMIC RESOLUTION 1TRO 13

REMARK 1 REF NATURE V. 335 321 1988 1TRO 14

REMARK 1 REFN ASTM NATUAS UK ISSN 0028-0836 006 1TRO 15

[..]

REMARK 2 1TRO 23

REMARK 2 RESOLUTION. 1.9 ANGSTROMS. 1TRO 24

REMARK 3 REFINEMENT. 1TRO 26

REMARK 3 PROGRAM PROLSQ 1TRO 27

REMARK 3 AUTHORS KONNERT,HENDRICKSON 1TRO 28

REMARK 3 R VALUE 0.167 1TRO 29

REMARK 3 RMSD BOND DISTANCES 0.015 ANGSTROMS 1TRO 30

REMARK 3 RMSD BOND ANGLES 1.3 DEGREES 1TRO 31

Following the introductory material, some specific information regarding the protein and its crystalline form are provided, including the protein sequence, secondary structure, disulfide bonds (where present) and the like. Also the dimensions of the unit cell, the space group and related information are given.


SEQRES 1 G 108 MET ALA GLN GLN SER PRO TYR SER ALA ALA MET ALA GLU 1TRO 106

SEQRES 2 G 108 GLU ARG HIS GLN GLU TRP LEU ARG PHE VAL ASP LEU LEU 1TRO 107

SEQRES 3 G 108 LYS ASN ALA TYR GLN ASN ASP LEU HIS LEU PRO LEU LEU 1TRO 108

[..]

FORMUL 13 HOH *572(H2 O1) 1TRO 124

HELIX 1 1A TYR A 7 GLN A 31 1 1TRO 125

HELIX 2 1B HIS A 35 MET A 42 1 1TRO 126

HELIX 3 1C ASP A 46 ARG A 63 1 1TRO 127

[..]

HELIX 23 4E ILE G 79 ALA G 91 1 1TRO 147

HELIX 24 4F VAL G 94 LEU G 105 1 1TRO 148

CRYST1 43.650 72.430 107.390 90.00 94.96 90.00 P 21 8 1TRO 149

Then come the actual atom coordinates (or structure), whose listing

takes up most of the average PDB file. Each listing begins with

"ATOM" and is followed by:

  • The atom number

  • The atom name (which is uniquely defined for each type of amino acid)

  • The type of residue

  • The chain identifier: In this case "A" indicating that this is

    one of several molecules in the model.

  • The residue's sequence number in the protein

  • The x, y and z coordinates

  • The occupancy (the fraction of atoms in the

    crystal that occupies the specified coordinates, usually one).

  • The temperature factor (or B factor), a measure for the coordinate uncertainty.Low B-factors ( 80) might indicate highly disordered parts of the structure or even mis-interpreted parts of the model.

  • And lastly, the models identifier (1TRO in this case) and the line number in the file).


ATOM 1 N SER A 5 4.462 27.337 23.235 1.00 61.04 1TRO 156

ATOM 2 CA SER A 5 5.077 28.626 22.900 1.00 62.14 1TRO 157

ATOM 3 C SER A 5 4.851 28.971 21.434 1.00 63.65 1TRO 158

ATOM 4 O SER A 5 4.235 28.222 20.647 1.00 65.52 1TRO 159

ATOM 5 CB SER A 5 6.545 28.668 23.270 1.00 58.51 1TRO 160

ATOM 6 OG SER A 5 7.320 27.704 22.589 1.00 50.29 1TRO 161

This goes on for a while, until the end of the peptide chain, which is marked by the "TER" line. If there are any other molecules that co-crystallized with the protein (such as solvent molecules or ligands) they are listed as "hetero-atoms" near the end of the file.


TER 4039 C J 19 1TRO4194

HETATM 4814 O HOH 1 20.236 5.712 14.585 1.00 49.26 1TRO4969

HETATM 4815 O HOH 2 30.581 1.151 18.292 1.00 29.71 1TRO4970

HETATM 4816 O HOH 3 28.781 -11.725 8.943 1.00 51.99 1TRO4971

HETATM 4817 O HOH 4 9.273 -10.253 29.757 1.00 56.19 1TRO4972

x-ray crystallography

The main technique for determining protein structures is x-ray

crystallography. Since the first protein structure (myoglobin) was solved by this

technique by John Kendrew and Max Perutz in the late fifties, several

thousand others followed. As can be appreciated from the picture on the right,

which shows John Kendrew with the structural model of myoglobin, at

that time the determination of a structure the size of a protein,

without the aid of a computer, was a formidable task.


It is important to note that in both x-ray crystallography and NMR,

protein structures are not measured directly in the

experiment. Rather, a set of data is collected (a diffraction pattern

or a NMR spectrum), from which a model of the protein structure

is derived. To appreciate the difference between data and structure,

we'll now look at two different structures of the same protein, and

the corresponding x-ray crystallographic data. For this, we will

concentrate on the bacterial light driven proton pump

bacteriorhodopsin. Click

here for more background information on bR.


First download two bR structures from the Protein Data Bank, with PDB entries

1BRR and 1QHJ. Save both PDB files to your local account (see the last

lecture if you forgot how to download from the PDB).

View the structures with rasmol:

rasmol 1BRR.pdb

to focus only on one of the three protein molecules in the file type

(in rasmol):

restrict *a

to see the main features of the structure:

cartoons

color structure

To highlight the dye (the light sensor in the protein interior):

select ret and *a

cpk

Have a close look at the structure, before repeating the procedure for 1QHJ.pdb

in a different window, such that you can compare the two structures.


Question:

By looking at the structures, which of the two structures would you

prefer, in terms of coordinate accuracy?


Remember, so far we only looked at the coordinates, which represent a model

that was optimized against the actually measured data. So, let

us now have a look at the data. In x-ray crystallography, data are

collected by measuring a diffraction pattern that is obtained from

x-rays reflected by a protein crystal. As mentioned in the lecture,

this diffraction pattern itself does not suffice to determine the complete structure

since only the amplitudes of the diffracted waves were collected, not

their phases. In x-ray crystallography, however, there are a number of

tricks available (e.g. isomorphous replacement, molecular replacement)

but we will not go into that in detail here. What is important to

remember is that eventually, an atomic electron density map is obtained.


Question:

Why do primarily the electrons of a molecular sample contribute to the

diffraction of x-rays? answer.


Visit the Uppsala electron density server

to view the electron density map 1BRR. Enter the PDB code (1BRR), and

wait for the summary page to load. Several plots with information on

this structure are available. Feel free to browse around to check the

meaning of the individual plots. At the bottom of the summary, the

electron density viewer can be activated. Select the "Upssala viewer"

instead of the default "Astex viewer" and input that the map should be

centered around residue A83 before clicking "Go". After a while, a

java applet should appear with the electron density and the model

structure visible. Shift the map somewhat to focus on the side-chain

of residue Tyr 83 (by clicking on "center" and then in the atom that

should be centered, until you are in the six-membered aromatic ring).

Do you find the electron density for the aromatic

ring convincing? Now shift the focus to residue A35 (by entering "A35"

in the text field under "center" and then hitting "center"). How is

the fit between the model and the data here? To see the retinal, the

light sensor in the center of the protein, center on A999.

Now repeat the procedure for entry 1QHJ. How is the fit for residue

A35? and for A83? And the retinal (called A300 here)?


Question:

Based on the data and on the model structures, would you say there is

a large impact of the resolution of the data on the accuracy of the structural model?


The highest resolution x-ray crystallographic structures have a

resolution of approx. 0.8A or even somewhat better. To see an example

of such a dataset, look at the density for structure 1G6X, and center

around residue A23. Do you recognize the difference in appearance of

the Tyrosine ring?


Question:

Although the resolution of this structure is rather high, at 0.86A, the

hydrogen atoms (e.g. on the side chain of TYR23) are hardly

visible. Why is this? answer.


NMR

The other main technique for determining protein structures is NMR. In

contrast to x-ray crystallography, no crystals are required for an NMR

experiment. Rather, the structure is determined of the protein in

solution. Therefore, it has the advantage that the protein can be

studied in its native environment. On the other hand, the resolution

of an NMR structure is usually lower and there is a size limitation of

a few hundred amino acids for structure determination using NMR.


It would go beyond the scope of this course to explain the

NMR experiment in detail. We will therefore only briefly touch on the

experimental setup and then focus on the structure building and

refinement step based on the obtained data.

The NMR signal is recorded as a nuclear magnetic resonance spectrum of predominantly the

hydrogen atoms after the sample has been subjected to a (number of)

strong magnetic pulse(s). Mainly hydrogen atoms give rise to the

signal, because of the magnetic spin properties of the hydrogen

nucleus (a proton). The naturally occurring isotopes of the other

elements that are found in proteins, carbon (12C) and

oxygen (16O),

have a zero nuclear magnetic moment. Nitrogen (14N) does

have a non-zero magnetic moment, but can usually not be used in NMR,

for reasons that would go beyond the scope of this course to explain.

These elements, therefore, can only

be utilized in NMR experiments when chemically replaced by a specific

isotope, like 13C or 15N. The most structurally

relevant information is usually obtained from a so called NOESY

experiment (Nuclear Overhauser Enhancement SpectroscopY).

The Nuclear Overhauser Effect or Nuclear Overhauser Enhancement is the

change (enhancement) of the signal intensity from a given nucleus as a

result of exciting or saturating the resonance frequency of another

nucleus. Since this effect is distance-dependent, it can be used to

derive the distance between an interacting pair of protons. In

practice, protons closer than 6A apart can be identified this way.


Now, we will calculate a model of the structure of a small protein,

the B1 domain of protein G,

from the proton-proton distance information obtained from a NOESY

experiment. Download the data file containing the distance information

here. You can have a look at the file

(with the program "more" or "less" or a browser or editor of your

choice) to assure yourself that there are indeed only distance bounds

listed in this file. Additionally, we need an initial guess of the

structure. Since we don't know the structure yet, we have to start from

an unstructured peptide chain, which can be obtained here . Have a look at the structure with:

rasmol proteinG.pdb

Finally, we need something called a

molecular topology, a chemical description of the protein: which atoms

does the molecule contain, which atoms are covalently bonded to each

other, etc. This molecular topology file is available here . Now, in principle, we have all the data

to attempt to build a structure that is in agreement with all

experimentally determined distances. The only thing we still need is

an input file for the

CNS program.


Note that, in contrast to x-ray crystallography, where a

single structure is presented, to reflect the fact that the NMR experiment probes

an ensemble of protein molecules in solution, an NMR structure is

usually represented by an ensemble of structures, that all fulfill the

NMR data. Start CNS with

tcsh

source /usr/global/cns/cns_solve_env

cns

10 structures of the B1 domain of protein G will now be calculated by

simulated annealing. This is a computationally intensive calculation,

as the structure is slowly, dynamically transformed from the extended

starting conformation to the real structure, by a slow-cooling

simulation, also called simulated annealing. As the calculation is

running, we can have a look at how exactly such a calculation proceeds,

and how the final structure is generated from the starting

guess. Download this file, open another shell, and

open the just downloaded file in pymol:

/usr/global/whatif/pymol/pymol sa.pdb

on the white bar, in the top pymol window, type:

show cartoon

and then press the "play" button at the bottom right of the main pymol

window, to see an animation of the simulated annealing structure

calculation procedure. If the movie plays too fast, on the menu, under

movie -> speed, choose a different speed.


When the CNS structure calculation has finished, switch back to that

window and type:

cat anneala_*.pdb > anneal.pdb

to combine all ten generated structures into one file. View the result

with:

rasmol anneal.pdb


Question:

Which parts of the structure are well-defined, and which parts show

more ambiguity?

There is also a x-ray structure available of the B1 domain of protein

G, available under the PDB code 1PGB. Download it from the Protein Data Bank and compare it to

the just calculated NMR structure.


Question:

What are the main differences between the NMR and x-ray structures of

the B1 domain of protein G? hint

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