4 Experimental Protein Structure Determination 2014 · Protein Structure Determination Using NMR spectroscopy 33 After this lesson you should be able to: • Analyze an NMR structure,

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Programme 8.00-8.15 Good morning and quiz results 8.15-8.20 Break 8.20-8.50 Experimental structure determination – part I 8.50-9.00 Break

9.00-9.30 Experimental structure determination – part II 9.30-9.45 Break 9.45-11.00 Exercise: Reading protein structure papers 11.00-11.30 Summary & Discussion 11.30-12.00 Quiz!

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Feedback Persons

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http://www.bio-evaluering.dk

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Experimental Protein Structure Determination

X-ray crystallography &

NMR Spectroscopy

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Learning Objectives

After today you should be able to:

•  Give an outline of the most important steps (and obstacles) in protein structure determination by X-ray crystallography and NMR spectroscopy.

•  Identify relevant parameters for evaluating the quality of protein structures determined by X-ray crystallography and NMR spectroscopy.

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Part I

X-ray crystallography

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Outline

•  Protein X-ray Crystallography – X-rays and diffraction – Crystallisation – The phase problem and solutions – Model refinement – Key parameters – Bottlenecks!

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Growing Crystals

•  Hanging drop technique (vapour diffusion)

•  Protein drop slowly equilibrates with reservoir solution leading to slow supersaturation and precipitation of the protein.

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X-Ray Sources

•  Synchrotron

ESRF

Grenoble, France

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X-rays

Fourier transform

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Recording Data

•  The Diffraction Experiment

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Object and Diffraction Image

•  Diffraction is a Fourier transform of the object (phase colouring)

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Diffraction Image

Structure factor Individual reflections

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Resolution rings

€

F hkl( ) = f j exp 2πi hx j + ky j + lz j( )[ ]j=1

N

∑

Reflection(s) €

Ihkl ∝ Fobs(hkl)2

The Phase Problem

•  Loss of phase information when recording data.

•  For crystals

Diffraction experiment

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Ihkl ∝ Fobs(hkl)2

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Phase Information

•  Our Fourier friends •  Phases switched

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From Data to Structure

•  Phasing methods –  MIR: multiple isomorphous replacements

•  Depends on scattering contribution from few heavy atoms, crystals must be isomorphous (same shape)

–  MAD (SAD): multiple (single) anomalous diffraction •  Depends on scattering from atoms above and below absorption

wavelenght •  Analogous to MIR

•  Molecular replacement –  “Stealing” the phases from a (very) similar structure to

phase data of homologous structure.

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The Importance of Resolution

high

low 4 Å

2 Å

3 Å

1 Å

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Phase Information

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Missing Data

•  Data completeness •  Simulated oscillation data with crystal decay

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•  Unit cell contents

Crystal Contents vs. Structure •  Lattice and unit cell

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Crystal Contents vs. Structure •  Lattice and unit cell •  Unit cell contents

•  Repeated in all three dimensions by translation

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Crystal Symmetry •  Asymmetric unit

–  Generates the unit cell by symmetry.

–  Usually one or more molecules/subunits.

•  Biological unit –  Biomolecule entry in

pdb file. –  May be more, less or

same as asym. unit.

•  Symmetry and symbols –  Examples:

P21,P43212, C222 –  Numbers refer to

rotation axes.

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Asymmetric Unit vs. Crystal

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Non-Crystallographic Symmetry •  NCS arises when the

symmetry of multimers does NOT coincide with crystal symmetry.

•  Useful in refinement of low-resolution structures –  Constraint (=) –  Restraint (~)

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Solvent in Protein Structures

Two levels of solvent description: •  Bulk solvent in solvent channels

–  Implicit description –  Not part of the final model, but part

of the data processing/handling

•  Crystallographic solvent –  Visible in electron density maps

•  Found on the surface and especially inside proteins.

•  Integral part of protein structure! –  Explicit atomic description

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B-factors •  Measure of atomic vibrations

<u2> is mean square displacement

•  Also contains contributions from: –  Crystal defects/decay –  Data incompleteness –  Multiple conformations –  Model/refinement errors –  Occupancy

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€

B = 8π u2

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Model Refinement

•  Model refinement consists of several rounds of simultaneous

–  Positional refinement –  B factor refinement –  Addition of water molecules –  Assignment of multiple

conformations

•  Goal: To minimize the difference between observed data and model.

•  Measure: R-factor

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Model vs. Data Agreement

•  R-factor:

•  Rfree: – Unbiased measure. – Calculated on 5-10%

of data not included in refinement.

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€

R =Fobs − Fcalc∑

Fobs∑

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Key Parameters •  Resolution •  R values

–  Agreement between data and model. –  Usually between 0.15 and 0.25, should not

exceed 0.30. •  R ~ Resolution / 10 •  R + 0.05 > Rfree > R.

•  Ramachandran plot

•  B factors –  Contributions from static and dynamic disorder

•  Well determined ~10-20 Å2, intermediate ~20-30 Å2, flexible 30-50 Å2, invisible >60 Å2.

Bottlenecks

•  Getting the protein in sufficient quantity and purity

•  Crystallisation (trial and error)

•  Diffraction/resolution limit

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Part II

NMR Spectroscopy

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Protein Structure Determination Using

NMR spectroscopy

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After this lesson you should be able to: •  Analyze an NMR structure, evaluate the over-all quality and

find well-defined and less defined regions of the structure

Outline •  A short introduction to the technique •  What is NMR be used for? •  From protein to spectra to structure •  A comparison to X-ray crystallography •  How to evaluate NMR structures?

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Learning Objectives & Outline

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NMR Basics

•  NMR is nuclear magnetic resonance

•  NMR spectroscopy is done on proteins IN SOLUTION

•  Only atoms 1H, 13C, 15N (and 31P) can be detected in NMR experiments

•  Proteins up to 30 kDa

•  Proteins stable at high concentration (0.5-1 mM), preferably @ room temperature

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What Is NMR Used For ?

•  Structural information

•  Studies of protein folding

•  Mapping interaction sites

•  Dynamics of proteins in solution

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NMR Structural Genomics Projects

•  Explore structure space

•  Help drug-design

•  Facilitate protein structure prediction

•  Development of newer more efficient and less time-consuming NMR techniques

Example: Structural genomics of the SARS-virus genome

1YSY, Peti et al, JVI, 2005

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The Basics of Data Collection

•  In a magnetic field the nuclei align like small magnets •  The nuclei are excited by radio frequency (RF) pulses •  When the RF pulse stops, the nuclei relax by (re-)emitting RF

radiation •  The measured emitted RF radiation is the NMR data •  It is Fourier-transformed into a spectrum

1H ppm 38

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NMR Spectrometers

The resolution of the spectra is depending on the strength of the magnetic field.

500-900 Mhz magnetic fields are used for protein NMR spectroscopy.

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•  The signal is seen as “peaks” in spectra.

•  Most hydrogen atoms in a folded protein have a unique frequency which is called chemical shift and is measured in ppm.

1H ppm

15N ppm

How Can an NMR Spectrum Give Information About the Structure?

1H,15N -HSQC

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Summary of the Technique •  Proteins in solution •  Only 13C,15N and 1H atoms are observed •  The nuclei are aligned in a magnetic field •  Exciting nuclei using RF-radiation •  Measure signal of emitted RF-radiation •  Fourier transform into spectrum •  Chemical environment influences the chemical shift of the

nuclei •  High magnetic strength = high resolution of spectra

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Magnetization transfer between atoms in space (NOESY spectra)

Magnetization transfer in 1H,15N NOESY

NMR Experiments for Structure Determination

Magnetization transfer between atoms which are chemically bonded (ex. COSY,1H,15N -HSQC, HNCACB)

Magnetization transfer in HNCACB

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•  NOE (Nuclear Overhauser Effect) •  The distance (H-H) is measured by the intensity (volume) of

the NOE peaks in the NMR spectrum and used as a RANGE

•  The NOESY quality => number of constraints •  The NOESY quality depends on:

–  Overlap of peaks in spectra –  Molecular rotation (tumbling) time –  Local (loop) dynamics

Distance Constraints from NOESY Spectra

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Different Classes of NOEs

•  Intra-residual (|i-i| = 0) •  Sequential (|i-j| = 1) •  Middle range (1<|i-j|<=5) •  Long range (|i-j|> 5)

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Other Constraints Used in NMR Structure Determination of Proteins

•  Dihedral angle restraints (phi, psi, chi) –  Ranges of dihedral angles based on

chemical shifts and j-coupling

•  Hydrogen bonds –  The NOE pattern can indicate hydrogen bonds

–  Slow exchange rates indicate hydrogen bonds

•  Residual dipolar couplings (RDCs) –  Constraints of the orientation of e.g. helical elements

relative to each other. 45

Distance Constraints

•  Protein –  Grey –  White –  Blue –  Red

•  Distance

constraints –  Green –  Yellow –  Cyan

Creator Dr. Shauna Farr-Jones

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Structure Calculation

•  All constraints are used as input in a program for protein structure calculation (X-plor, Cyana, CNS)

•  The programs are based on energy terms

•  Programs include energy terms for general geometrical features of proteins (bond lengths, angles etc.)

•  The total energy is a sum of the energy based on the constraints and the ideal geometry terms

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Energy Minimization

•  The total energy is minimized to get representations of the structure which obey geometrical (chemical) and experimental constraints

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Iterations Between Structure Calculation and Interpretation of Spectra

•  Typically 100-200 structures are calculated

•  VIOLATIONS = constraints that are not fulfilled

•  Correction for wrong assignments

•  Some spectroscopists just remove violated constraints….

“Consistently violated restraints were identified and eliminated in subsequent rounds of structure calculations until a consistent set of restraints was obtained with few violations in the ensemble. “

Keeler C et al. JMB, 2003

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Deposited NMR Structure

•  Finally, an ensemble of the 10-25 structures with lowest total energy and lowest number of violations are deposited in the Protein Data Bank

•  The restraints are deposited in the BioMagResBank (BMRB)

•  The ensemble shows possible structures within the space defined by the constraints

The ensemble of 20 NMR structures of the U-box domain from Atpub14 deposited in PDB (1T1H)

Andersen et al. , JBC, 2004

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Summary of NMR Structure Determination

•  Assign peaks •  Assign NOESY spectra •  Obtain other restraints (RDCs, angles) •  Structure calculation •  Check violations/ bad assignments •  Deposit structure + restraints

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NMR Spectroscopy vs. X-ray Crystallography

•  Hydrogen atoms are observed! •  Only 13C,15N and 1H are observed •  Study of proteins in solution •  Only proteins up to 30-40 kDa •  No total “map” of the structure •  Information used is incomplete and used as restraints •  An ensemble of structures is submitted to PDB •  The solved structure can be used for further dynamics

characterization with NMR

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How to Evaluate NMR Structures?

•  What types of experiments are used?

– Homo-nuclear (COSY, NOESY, TOCSY)

– Hetero-nuclear (HSQC, HNCACB, HCCH-TOCSY)

Homo-nuclear Bad resolution of spectra

=> Best for proteins < 10 kDA

Hetero-nuclear Better resolution of spectra

=> Better in general

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Well-defined structures RMSDs < 0.6 Å

•  Atomic backbone RMSD

Less well-defined structures RMSDs > 0.6 Å 3GF1, Cooke et al. Biochemistry, 1991 1T1H, Andersen et al. JBC, 2004

€

RMSD =xi − xi

'( )2

1

n∑

n


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•  What types of constraints? •  Numbers of constraints?

• Distance restraints

• Angle restraints

• RDCs

• Hydrogen bonds

In well-defined regions each residues have at least 20 restraints

Best if no violations of distance restraints >0.5Å

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Which regions in the structure are most well-defined?

Look at the pdb ensembles to see which regions are well-defined

1RJH

Nielbo et al, Biochemistry, 2003


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•  What regions in the structure are most well-defined? Look at restraints in PDB (Experimental Method à data)

Example of restraints ASSIGN (RESID 252 AND NAME HA ) (RESID 260 AND NAME HD# ) 3.38 1.58 1.58 !

ASSIGN (RESID 252 AND NAME HA ) (RESID 261 AND NAME HE# ) 3.23 1.44 1.44 !

ASSIGN (RESID 252 AND NAME HA ) (RESID 307 AND NAME HD# ) 3.56 1.77 1.77 !

ASSIGN (RESID 252 AND NAME HA ) (RESID 307 AND NAME HG ) 3.65 1.85 1.85 !

ASSIGN (RESID 252 AND NAME HA ) (RESID 311 AND NAME HG2# ) 3.65 1.85 1.85 !

ASSIGN (RESID 252 AND NAME HA ) (RESID 252 AND NAME HD# ) 2.96 1.17 1.17 !

ASSIGN (RESID 252 AND NAME HA ) (RESID 252 AND NAME HE# ) 3.65 1.85 1.85 !

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A Word of Caution •  RMSD depends on:

–  Number of structures in ensemble (PDB file).

–  Total number of structures generated

•  Ensemble RMSD should be maximized within the experimental restraints, NOT minimized!

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Spronk 2003, Journal of Biomolecular NMR

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Summary on Evaluation

•  Homo-/hetero-nuclear NMR? •  RMSD (be careful!)? •  Types of constraints? •  Numbers of constraints? •  Violations? •  Specific regions?

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Summary: X-rays vs. NMR

§  X-ray crystallography §  X-rays

§  Proteins of any size §  Proteins in crystal §  Complete data/total

map of structure §  Many details – one

model §  Resolution, R-values,

Ramachandran plot

§  NMR spectroscopy §  Radio waves +

magnetic field §  Proteins below 50 kDa §  Proteins in solution §  Incomplete data

§  Fewer details – many models (ensemble)

§  Restraint violations, RMSD, Ramachandran plot

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Documents

4 Experimental Protein Structure Determination 2014 · Protein Structure Determination Using NMR spectroscopy 33 After this lesson you should be able to: • Analyze an NMR structure,